G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
cyclin-dependent kinase 5
G00000167 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000023920 (Vega human gene)
ENSG00000164885 (Ensembl human gene)
1020 (Entrez Gene)
498 (G2Cdb plasticity & disease)
CDK5 (GeneCards)
123831 (OMIM)
Marker Symbol
HGNC:1774 (HGNC)
Protein Sequence
Q00535 (UniProt)

Synonyms (1)


Literature (145)

Pubmed - other

  • Genetic susceptibility to distinct bladder cancer subphenotypes.

    Guey LT, García-Closas M, Murta-Nascimento C, Lloreta J, Palencia L, Kogevinas M, Rothman N, Vellalta G, Calle ML, Marenne G, Tardón A, Carrato A, García-Closas R, Serra C, Silverman DT, Chanock S, Real FX, Malats N and EPICURO/Spanish Bladder Cancer Study investigators

    Spanish National Cancer Research Centre, Madrid, Spain.

    Background: Clinical, pathologic, and molecular evidence indicate that bladder cancer is heterogeneous with pathologic/molecular features that define distinct subphenotypes with different prognoses. It is conceivable that specific patterns of genetic susceptibility are associated with particular subphenotypes.

    Objective: To examine evidence for the contribution of germline genetic variation to bladder cancer heterogeneity.

    The Spanish Bladder Cancer/EPICURO Study is a case-control study based in 18 hospitals located in five areas in Spain. Cases were patients with a newly diagnosed, histologically confirmed, urothelial cell carcinoma of the bladder from 1998 to 2001. Case diagnoses were reviewed and uniformly classified by pathologists following the World Health Organisation/International Society of Urological Pathology 1999 criteria. Controls were hospital-matched patients (n=1149).

    Measurements: A total of 1526 candidate variants in 423 candidate genes were analysed. Three distinct subphenotypes were defined according to stage and grade: low-grade nonmuscle invasive (n=586), high-grade nonmuscle invasive (n=219), and muscle invasive (n=246). The association between each variant and subphenotype was assessed by polytomous risk models adjusting for potential confounders. Heterogeneity in genetic susceptibility among subphenotypes was also tested.

    Two established bladder cancer susceptibility genotypes, NAT2 slow-acetylation and GSTM1-null, exhibited similar associations among the subphenotypes, as did VEGF-rs25648, which was previously identified in our study. Other variants conferred risks for specific tumour subphenotypes such as PMS2-rs6463524 and CD4-rs3213427 (respective heterogeneity p values of 0.006 and 0.004), which were associated with muscle-invasive tumours (per-allele odds ratios [95% confidence interval] of 0.56 [0.41-0.77] and 0.71 [0.57-0.88], respectively) but not with non-muscle-invasive tumours. Heterogeneity p values were not robust in multiple testing according to their false-discovery rate.

    Conclusions: These exploratory analyses suggest that genetic susceptibility loci might be related to the molecular/pathologic diversity of bladder cancer. Validation through large-scale replication studies and the study of additional genes and single nucleotide polymorphisms are required.

    Funded by: Intramural NIH HHS: ZIA CP010136-16

    European urology 2010;57;2;283-92

  • Genetic variants of cyclin-dependent kinase 5 regulatory subunit associated protein 1-like 1 and transcription factor 7-like 2 are not associated with polycystic ovary syndrome in Chinese women.

    Liu X, Li L, Chen ZJ, Lu Z, Shi Y and Zhao Y

    Key Laboratory for ReproductiveMedicine of Shandong Province, Shandong Provincial Hospital, Shandong University, Jinan, Shandong, People's Republic of China.

    Polycystic ovary syndrome (PCOS) is a common disorder in women that shares many genetic features with type 2 diabetes mellitus. Novel risk loci for type 2 diabetes, single nucleotide polymorphism (SNP) rs7756992 in cyclin-dependent kinase 5 (CDK5) regulatory subunit associated protein 1-like 1 (CDKAL1), rs290487 and rs11196218 in transcription factor 7-like 2 (TCF7L2), were recently identified. The aim of this study was to test whether these loci are also associated with PCOS. We recruited 826 patients with PCOS and 620 healthy controls for case-control analysis. The genotypes of these three SNPs were identified. The relationships between PCOS-related clinical endocrine and metabolic features and genotypes were also analyzed. Genotype distribution of these three loci in case and control groups showed no deviation from the Hardy-Weinberg equilibrium. No significant differences in genotype and allele frequencies were found between patients with PCOS and healthy controls. No associations were observed between genotypes of the three SNPs and the clinical endocrine and metabolic features of patients with PCOS in case group after adjustment for body mass index. We concluded that rs7756992 in CDKAL1, rs290487 and rs11196218 in TCF7L2 have no associations with PCOS or PCOS-related clinical features.

    Gynecological endocrinology : the official journal of the International Society of Gynecological Endocrinology 2010;26;2;129-34

  • Cdk5-dependent regulation of Rho activity, cytoskeletal contraction, and epithelial cell migration via suppression of Src and p190RhoGAP.

    Tripathi BK and Zelenka PS

    Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, 5635 Fishers Lane, Room 1127, Bethesda, MD 20892, USA.

    Cdk5 regulates adhesion and migration in a variety of cell types. We previously showed that Cdk5 is strongly activated during stress fiber formation and contraction in spreading cells. Here we determine the mechanism linking Cdk5 to stress fiber contractility and its relevance to cell migration. Immunofluorescence showed that Cdk5 colocalized with phosphorylated myosin regulatory light chain (pMRLC) on contracting stress fibers. Inhibiting Cdk5 activity by various means significantly reduced pMRLC level and cytoskeletal contraction, with loss of central stress fibers. Blocking Cdk5 activity also reduced Rho-Rho kinase (ROCK) signaling, which is the principal pathway of myosin phosphorylation under these conditions. Next, we examined the effect of Cdk5 activity on Src, a known regulator of Rho. Inhibiting Cdk5 activity increased Src activation and phosphorylation of its substrate, p190RhoGAP, an upstream inhibitor of Rho. Inhibiting both Cdk5 and Src activity completely reversed the effect of Cdk5 inhibition on Rho and prevented the loss of central stress fibers, demonstrating that Cdk5 exerts its effects on Rho-ROCK signaling by suppressing Src activity. Moreover, inhibiting either Cdk5 or ROCK activity increased cell migration to an equal extent, while inhibiting both kinases produced no additional effect, demonstrating that Cdk5-dependent regulation of ROCK activity is a physiological determinant of migration rate.

    Funded by: Intramural NIH HHS; NEI NIH HHS: Z01 EY000238

    Molecular and cellular biology 2009;29;24;6488-99

  • PTEN identified as important risk factor of chronic obstructive pulmonary disease.

    Hosgood HD, Menashe I, He X, Chanock S and Lan Q

    Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.

    Common genetic variation may play an important role in altering chronic obstructive pulmonary disease (COPD) risk. In Xuanwei, China, the COPD rate is more than twice the Chinese national average, and COPD is strongly associated with in-home coal use. To identify genetic variation that may be associated with COPD in a population with substantial in-home coal smoke exposures, we evaluated 1261 single nucleotide polymorphisms (SNPs) in 380 candidate genes potentially relevant for cancer and other human diseases in a population-based case-control study in Xuanwei (53 cases; 107 controls). PTEN was the most significantly associated gene with COPD in a minP analysis using 20,000 permutations (P=0.00005). SNP-based analyses found that homozygote variant carriers of PTEN rs701848 (OR(TT)=0.12, 95% CI=0.03-0.47) had a significant decreased risk of COPD. PTEN, or phosphatase and tensin homolog, is an important regulator of cell cycle progression and cellular survival via the AKT signaling pathway. Our exploratory analysis suggests that genetic variation in PTEN may be an important risk factor of COPD in Xuanwei. However, due to the small sample size, additional studies are needed to evaluate these associations within Xuanwei and other populations with coal smoke exposures.

    Funded by: Intramural NIH HHS: Z99 CA999999

    Respiratory medicine 2009;103;12;1866-70

  • No association of CDK5 genetic variants with Alzheimer's disease risk.

    Vázquez-Higuera JL, Mateo I, Sánchez-Juan P, Rodríguez-Rodríguez E, Infante J, Berciano J and Combarros O

    Neurology Service and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, University Hospital Marqués de Valdecilla (University of Cantabria), Santander, Spain. josevazquezhiguera@hotmail.com

    Background: As cyclin-dependent kinase 5 (CDK5) has been implicated in the abnormal hyperphosphorylation of tau in Alzheimer's disease (AD) brain, and the development of neurofibrillary tangles, we examined the contribution of this gene to the susceptibility for AD.

    Methods: We examined genetic variations of CDK5 by genotyping haplotype tagging SNPs (htSNPs) (rs9278, rs2069459, rs891507, rs2069454, rs1549759 and rs2069442) in a group of 408 Spanish AD cases and 444 controls.

    Results: There were no differences in the genotypic, allelic or haplotypic distributions between cases and controls in the overall analysis or after stratification by APOE epsilon4 allele.

    Conclusion: Our negative findings in the Spanish population argue against the hypothesis that CDK5 genetic variations are causally related to AD risk. Still, additional studies using different sets of patients and control subjects deserve further attention, since supporting evidence for association between CDK5 gene and AD risk in the Dutch population exists.

    BMC medical genetics 2009;10;68

  • [Deregulation of Cdk5 in neuronal death and neurodegenerative diseases].

    Saito T


    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 2009;54;7;820-4

  • [Functions of Cdk5 in non-neuronal tissues: focusing on regulation of insulin secretion].

    Tomizawa K


    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 2009;54;7;808-12

  • [Role of Cdk5 in dopamine signaling and addiction].

    Nishi A


    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 2009;54;7;813-9

  • Single-nucleotide polymorphisms in the promoter of the CDK5 gene and lung cancer risk in a Korean population.

    Choi HS, Lee Y, Park KH, Sung JS, Lee JE, Shin ES, Ryu JS and Kim YH

    Genomic Research Center for Lung and Breast/Ovarian Cancers, Korea University Anam Hospital, Seoul, Korea.

    Cyclin-dependent kinase 5 (CDK5), a proline-directed serine/threonine kinase, which was originally known for its regulatory role in neuronal activities, has recently been suggested to play a role in extraneuronal activities. For example, a recent study detected overexpression of the CDK5 gene in non-small-cell lung cancer. Therefore, in order to explore the association of the CDK5 gene with lung cancer risk in a Korean population, the genotypes of the CDK5 promoter region were determined in 407 lung cancer patients and 402 normal participants. The result showed that the -904 G>A genotype affected susceptibility to lung cancer risk (odd ratios (OR)=1.53, 95% confidence interval (CI)=1.02-2.32). Furthermore, subsequent haplotype analysis of three single-nucleotide polymorphism (SNP) regions suggested that the A-G-C haplotype was associated with a higher overall risk of lung cancer (OR=1.59, 95% CI=1.16-2.18). These results suggest that CDK5 promoter polymorphisms contribute to the genetic susceptibility to lung cancer in the Korean population.

    Journal of human genetics 2009;54;5;298-303

  • Combined kinase inhibition modulates parkin inactivation.

    Rubio de la Torre E, Luzón-Toro B, Forte-Lago I, Minguez-Castellanos A, Ferrer I and Hilfiker S

    Institute of Parasitology and Biomedicine López-Neyra, Consejo Superior de Investigaciones Científicas, Granada, Spain.

    Mutations in the parkin gene cause autosomal-recessive, juvenile-onset parkinsonism, and parkin dysfunction may also play a role in the pathogenesis of sporadic Parkinson disease (PD). Although its precise function remains largely unknown, parkin seems to play a neuroprotective role. Several studies indicate that changes in parkin solubility induced by post-translational modifications, such as S-nitrosylation or dopamine modification, comprise one mechanism of parkin inactivation associated with disease. Protein phosphorylation events have recently been linked to the molecular mechanism(s) underlying PD, but the role of this post-translational modification for parkin function has remained unclear. Here we report that compound phosphorylation of parkin by both casein kinase I and cyclin-dependent kinase 5 (cdk5) decreases parkin solubility, leading to its aggregation and inactivation. Combined kinase inhibition partially reverses the aggregative properties of several pathogenic point mutants in cultured cells. Enhanced parkin phosphorylation is detected in distinct brain areas of individuals with sporadic PD and correlates with increases in the levels of p25, the activator of cdk5. These findings indicate that casein kinase I and cdk5 may represent novel combinatorial therapeutic targets for treating PD.

    Human molecular genetics 2009;18;5;809-23

  • The role of Cdk5 in retinoic acid-induced apoptosis of cervical cancer cell line.

    Kuo HS, Hsu FN, Chiang MC, You SC, Chen MC, Lo MJ and Lin H

    Department of Life Sciences, National Chung Hsing University, Taichung 402, Taiwan, Republic of China.

    Cdk5 is a small serine/threonine protein kinase which belongs to Cdk family. Unlike other Cdk members, so far Cdk5 is known to be irrelevant in cell cycle. Cdk5 kinase activity is regulated by binding with its activator, p35. Our previous results indicate that CdkS and p35 are involved in drugs-induced apoptosis of prostate cancer cells. Retinoic acid (RA) is one of the vitamin A-related compounds. Because of its potency on biological functions, it has been widely studied in its novel actions including the ability to inhibit cancer cell growth and to induce apoptosis. Here, we report that RA treatment decreased the growth of human cervical cancer cell line, HeLa, and Cdk5 contributed to this effect. The involvement of Cdk5 in RA-reduced cell survival was performed by treatments of Cdk5 inhibitor and siRNA. We further identified that RA-induced growth inhibition was partly correlated to Cdk5 activity-related apoptosis by detecting cell cycle distribution of sub G1 phase and the signals of Annexin V staining. In addition, our results also indicated that Cdk5 activity was involved in RA-induced HeLa apoptosis by detecting cleavages of caspase-3 and its substrate, PARP (poly (ADP-ribose) polymerases) Interestingly, the nuclear localizations of Cdk5 and p35 proteins were increased by RA treatment, which, again, suggests the involvement of Cdk5 and p35 in RA-induced apoptotic effects. In conclusion, we provide evidence to suggest that Cdk5 and p35 might play important roles in RA-induced HeLa apoptosis.

    The Chinese journal of physiology 2009;52;1;23-30

  • Cdk5 and the non-catalytic arrest of the neuronal cell cycle.

    Zhang J and Herrup K

    Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082, USA.

    Cyclin-dependent kinase 5 (Cdk5) is a nontraditional Cdk that is primarily active in postmitotic neurons. An important core function of Cdk5 involves regulating the migration and maturation of embryonic post-mitotic neurons. These developmental roles are dependent on its kinase activity. Initially, there was little evidence indicating a role for Cdk5 in normal cell cycle regulation. Recent data from our lab, however, suggest that Cdk5 plays a crucial role as a cell cycle suppressor in normal post-mitotic neurons and neuronal cell lines. It performs this foundation in a kinase independent manner. Cdk5 normally found in both nucleus and cytoplasm, but it exits the nucleus in neurons risk to death in an AD patient's brain. The shift in sub-cellular location is accompanied by cell cycle re-entry and neuronal death. This "new" function of Cdk5 raises cautions in the design of Cdk5-directed drugs for the therapy of neurodegenerative diseases.

    Funded by: NIA NIH HHS: R01 AG024494; NINDS NIH HHS: R01 NS020591, R01 NS020591-25

    Cell cycle (Georgetown, Tex.) 2008;7;22;3487-90

  • Pathway-based evaluation of 380 candidate genes and lung cancer susceptibility suggests the importance of the cell cycle pathway.

    Hosgood HD, Menashe I, Shen M, Yeager M, Yuenger J, Rajaraman P, He X, Chatterjee N, Caporaso NE, Zhu Y, Chanock SJ, Zheng T and Lan Q

    Occupational and Environmental Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. hosgoodd@mail.nih.gov

    Common genetic variation may play an important role in altering lung cancer risk. We conducted a pathway-based candidate gene evaluation to identify genetic variations that may be associated with lung cancer in a population-based case-control study in Xuan Wei, China (122 cases and 111 controls). A total of 1260 single-nucleotide polymorphisms (SNPs) in 380 candidate genes for lung cancer were successfully genotyped and assigned to one of 10 pathways based on gene ontology. Logistic regression was used to assess the marginal effect of each SNP on lung cancer susceptibility. The minP test was used to identify statistically significant associations at the gene level. Important pathways were identified using a test of proportions and the rank truncated product methods. The cell cycle pathway was found as the most important pathway (P = 0.044) with four genes significantly associated with lung cancer (PLA2G6 minP = 0.001, CCNA2 minP = 0.006, GSK3 beta minP = 0.007 and EGF minP = 0.013), after adjusting for multiple comparisons. Interestingly, most cell cycle genes that were associated with lung cancer in this analysis were concentrated in the AKT signaling pathway, which is essential for regulation of cell cycle progression and cellular survival, and may be important in lung cancer etiology in Xuan Wei. These results should be viewed as exploratory until they are replicated in a larger study.

    Funded by: Intramural NIH HHS; NCI NIH HHS: TU2 CA105666

    Carcinogenesis 2008;29;10;1938-43

  • Cooperative roles of c-Abl and Cdk5 in regulation of p53 in response to oxidative stress.

    Lee JH, Jeong MW, Kim W, Choi YH and Kim KT

    Department of Life Science, Division of Molecular and Life Science, Systems-Biodynamics National Care Research Center, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea.

    The p53 tumor suppressor protein, a critical modulator of cellular stress responses, is activated through diverse mechanisms that result in its stabilization and transcriptional activation. p53 activity is controlled by transcriptional, translational, and post-translational regulation. The major mechanisms of p53 regulation occur primarily through interactions with HDM2, an E3 ubiquitin ligase that leads to p53 nuclear export and degradation. Here, we demonstrate that hydrogen peroxide-induced oxidative stress elicits down-regulation of HDM2. c-Abl mediates down-regulation of HDM2, leading to an increase of p53 level. Moreover, Cdk5 (cyclin-dependent kinase 5), a proline-directed Ser/Thr kinase, additionally increases p53 stability via post-translational modification of p53 in response to hydrogen peroxide. The p53 protein stabilized by c-Abl and Cdk5 is transcriptionally active; however, transcription of its target gene is differentially regulated with selective binding of p53 on promoter regions of its target genes by c-Abl. In addition, c-Abl modulates Cdk5 activity via phosphorylation of tyrosine 15 in cooperation with cleavage of p35 to p25. Our results show that c-Abl and Cdk5 cooperatively regulate maximal activation of p53, resulting in neuronal death in response to oxidative stress by hydrogen peroxide. These findings aid in clarifying the mechanism underlying the occurrence of neuronal apoptosis as a result of c-Abl and Cdk5-mediated p53 stabilization and transcriptional activation.

    The Journal of biological chemistry 2008;283;28;19826-35

  • Impact of nine common type 2 diabetes risk polymorphisms in Asian Indian Sikhs: PPARG2 (Pro12Ala), IGF2BP2, TCF7L2 and FTO variants confer a significant risk.

    Sanghera DK, Ortega L, Han S, Singh J, Ralhan SK, Wander GS, Mehra NK, Mulvihill JJ, Ferrell RE, Nath SK and Kamboh MI

    Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. Dharambir-Sanghera@ouhsc.edu

    Background: Recent genome-wide association (GWA) studies have identified several unsuspected genes associated with type 2 diabetes (T2D) with previously unknown functions. In this investigation, we have examined the role of 9 most significant SNPs reported in GWA studies: [peroxisome proliferator-activated receptor gamma 2 (PPARG2; rs 1801282); insulin-like growth factor two binding protein 2 (IGF2BP2; rs 4402960); cyclin-dependent kinase 5, a regulatory subunit-associated protein1-like 1 (CDK5; rs7754840); a zinc transporter and member of solute carrier family 30 (SLC30A8; rs13266634); a variant found near cyclin-dependent kinase inhibitor 2A (CDKN2A; rs10811661); hematopoietically expressed homeobox (HHEX; rs 1111875); transcription factor-7-like 2 (TCF7L2; rs 10885409); potassium inwardly rectifying channel subfamily J member 11(KCNJ11; rs 5219); and fat mass obesity-associated gene (FTO; rs 9939609)].

    Methods: We genotyped these SNPs in a case-control sample of 918 individuals consisting of 532 T2D cases and 386 normal glucose tolerant (NGT) subjects of an Asian Sikh community from North India. We tested the association between T2D and each SNP using unconditional logistic regression before and after adjusting for age, gender, and other covariates. We also examined the impact of these variants on body mass index (BMI), waist to hip ratio (WHR), fasting insulin, and glucose and lipid levels using multiple linear regression analysis.

    Results: Four of the nine SNPs revealed a significant association with T2D; PPARG2 (Pro12Ala) [odds ratio (OR) 0.12; 95% confidence interval (CI) (0.03-0.52); p = 0.005], IGF2BP2 [OR 1.37; 95% CI (1.04-1.82); p = 0.027], TCF7L2 [OR 1.64; 95% CI (1.20-2.24); p = 0.001] and FTO [OR 1.46; 95% CI (1.11-1.93); p = 0.007] after adjusting for age, sex and BMI. Multiple linear regression analysis revealed significant association of two of nine investigated loci with diabetes-related quantitative traits. The 'C' (risk) allele of CDK5 (rs 7754840) was significantly associated with decreased HDL-cholesterol levels in both NGT (p = 0.005) and combined (NGT and T2D) (0.005) groups. The less common 'C' (risk) allele of TCF7L2 (rs 10885409) was associated with increased LDL-cholesterol (p = 0.010) in NGT and total and LDL-cholesterol levels (p = 0.008; p = 0.003, respectively) in combined cohort.

    Conclusion: To our knowledge, this is first study reporting the role of some recently emerged loci with T2D in a high risk population of Asian Indian origin. Further investigations are warranted to understand the pathway-based functional implications of these important loci in T2D pathophysiology in different ethnicities.

    Funded by: FIC NIH HHS: K01 TW006087, KO1 TW006087

    BMC medical genetics 2008;9;59

  • Novel genetic tools reveal Cdk5's major role in Golgi fragmentation in Alzheimer's disease.

    Sun KH, de Pablo Y, Vincent F, Johnson EO, Chavers AK and Shah K

    Department of Chemistry and Purdue Cancer Center, Purdue University, West Lafayette, IN 47907, USA.

    Golgi fragmentation is a common feature in multiple neurodegenerative diseases; however, the precise mechanism that causes fragmentation remains obscure. A potential link between Cdk5 and Golgi fragmentation in Alzheimer's disease (AD) was investigated in this study. Because Golgi is physiologically fragmented during mitosis by Cdc2 kinase and current Cdk5-specific chemical inhibitors target Cdc2 as well, development of novel tools to modulate Cdk5 activity was essential. These enzyme modulators, created by fusing TAT sequence to Cdk5 activators and an inhibitor peptide, enable specific activation and inhibition of Cdk5 activity with high temporal control. These genetic tools revealed a major role of Cdk5 in Golgi fragmentation upon beta-amyloid and glutamate stimulation in differentiated neuronal cells and primary neurons. A crucial role of Cdk5 was further confirmed when Cdk5 activation alone resulted in robust Golgi disassembly. The underlying mechanism was unraveled using a chemical genetic screen, which yielded cis-Golgi matrix protein GM130 as a novel substrate of Cdk5. Identification of the Cdk5 phosphorylation site on GM130 suggested a mechanism by which Cdk5 may cause Golgi fragmentation upon deregulation in AD. As Cdk5 is activated in several neurodegenerative diseases where Golgi disassembly also occurs, this may be a common mechanism among multiple disorders.

    Molecular biology of the cell 2008;19;7;3052-69

  • Relative resistance of Cdk5-phosphorylated CRMP2 to dephosphorylation.

    Cole AR, Soutar MP, Rembutsu M, van Aalten L, Hastie CJ, McLauchlan H, Peggie M, Balastik M, Lu KP and Sutherland C

    Neurosciences Institute, Division of Pathology and Neuroscience, University of Dundee, Ninewells Hospital, Dundee, Scotland, UK.

    Collapsin response mediator protein 2 (CRMP2) binds to microtubules and regulates axon outgrowth in neurons. This action is regulated by sequential phosphorylation by the kinases cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3) at sites that are hyperphosphorylated in Alzheimer disease. The increased phosphorylation in Alzheimer disease could be due to increases in Cdk5 and/or GSK3 activity or, alternatively, through decreased activity of a CRMP phosphatase. Here we establish that dephosphorylation of CRMP2 at the residues targeted by GSK3 (Ser-518/Thr-514/Thr-509) is carried out by a protein phosphatase 1 family member in vitro, in neuroblastoma cells, and primary cortical neurons. Inhibition of GSK3 activity using insulin-like growth factor-1 or the highly selective inhibitor CT99021 causes rapid dephosphorylation of CRMP2 at these sites. In contrast, pharmacological inhibition of Cdk5 using purvalanol results in only a gradual and incomplete dephosphorylation of CRMP2 at the site targeted by Cdk5 (Ser-522), suggesting a distinct phosphatase targets this residue. A direct comparison of dephosphorylation at the Cdk5 versus GSK3 sites in vitro shows that the Cdk5 site is comparatively resistant to phosphatase treatment. The presence of the peptidyl-prolyl isomerase enzyme, Pin1, does not affect dephosphorylation of Ser-522 in vitro, in cells, or in Pin1 transgenic mice. Instead, the relatively high resistance of this site to phosphatase treatment is at least in part due to the presence of basic residues located nearby. Similar sequences in Tau are also highly resistant to phosphatase treatment. We propose that relative resistance to phosphatases might be a common feature of Cdk5 substrates and could contribute to the hyperphosphorylation of CRMP2 and Tau observed in Alzheimer disease.

    Funded by: Biotechnology and Biological Sciences Research Council: C18727; Medical Research Council

    The Journal of biological chemistry 2008;283;26;18227-37

  • Cdk5-mediated phosphorylation of c-Myc on Ser-62 is essential in transcriptional activation of cyclin B1 by cyclin G1.

    Seo HR, Kim J, Bae S, Soh JW and Lee YS

    Division of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Korea.

    It has been reported previously that cyclin G1 enables cells to overcome radiation-induced G(2) arrest and increased cell death and that these effects are mediated by transcriptional activation of cyclin B1. In this study, we further investigated the mechanism by which cyclin G1 transcriptionally activates cyclin B1. Deletion or point mutations within the cyclin B1 promoter region revealed that the c-Myc binding site (E-box) is necessary for cyclin G1-mediated transcriptional activation of cyclin B1 to occur. In addition, the kinase activity of Cdk5 was increased by cyclin G1 overexpression, and Cdk5 directly phosphorylated c-Myc on Ser-62. Furthermore, cyclin G1 mediated increased radiosensitivity, and radiation-induced M phase arrest was attenuated when RNA interference of Cdk5 was treated. Taken together, the results of this study indicate that Cdk5 activation in cells that overexpress cyclin G1 leads to c-Myc phosphorylation on Ser-62, which is responsible for cyclin G1-mediated transcriptional activation of cyclin B1.

    The Journal of biological chemistry 2008;283;23;15601-10

  • Cdk5-mediated regulation of the PIKE-A-Akt pathway and glioblastoma cell invasion.

    Liu R, Tian B, Gearing M, Hunter S, Ye K and Mao Z

    Departments of Pharmacology, Neurology, and Pathology, Emory University School of Medicine, Atlanta, GA 30322, USA.

    Isoform A of phosphatidylinositol 3-kinase enhancer (PIKE-A) is a newly identified prooncogenic factor that has been implicated in cancer cell growth. How PIKE-A activity is regulated in response to growth signal is poorly understood. Here, we demonstrate that cyclin dependent kinase 5 (Cdk5), a protein known to function mainly in postmitotic neurons, directly phosphorylates PIKE-A at Ser-279 in its GTPase domain in glioblastoma cells. This phosphorylation event stimulates PIKE-A GTPase activity and the activity of its downstream effector Akt. Growth signal activates Cdk5 and results in a Cdk5-dependent accumulation of phosphorylated PIKE-A and activation of Akt in the nucleus. Furthermore, PIKE-A phosphorylation and Cdk5 are increased in human glioblastoma specimens. Phosphorylation of PIKE-A by Cdk5 mediates growth factor-induced migration and invasion of human glioblastoma cells. Together, these findings identify PIKE as the first Cdk5 target in cancer cells, revealing a previously undescribed regulatory mechanism that mediates growth signal-induced activation of PIKE-A/Akt and tumor invasion.

    Funded by: NIA NIH HHS: AG023695, R01 AG023695; NINDS NIH HHS: R01 NS048254

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;21;7570-5

  • Calmodulin binding and Cdk5 phosphorylation of p35 regulate its effect on microtubules.

    He L, Hou Z and Qi RZ

    Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.

    In the nervous system, Cdk5 and its neuronal activator p35 are involved in the control of various activities, including neuronal differentiation and migration. Recently, we have reported that p35 is a microtubule-associated protein that regulates microtubule dynamics ( Hou, Z., Li, Q., He, L., Lim, H. Y., Fu, X., Cheung, N. S., Qi, D. X., and Qi, R. Z. (2007) J. Biol. Chem. 282, 18666-18670 ). Here we present two regulatory modes of p35 function as a microtubule-associated protein. First, p35 is Ca(2+)-dependent calmodulin (CaM)-binding protein. The CaM- and microtubule binding domains are localized to overlapping regions at the N terminus of p35. Within the CaM-binding region, Ala substitution for Trp-52 abolishes the CaM-binding activity, corroborating specific CaM-binding of p35. Furthermore, CaM blocks p35 association with microtubules in a Ca(2+)-specific manner, suggesting that p35 may be involved in the Ca(2+)/CaM-mediated inhibition of microtubule assembly. Second, p35 phosphorylation by Cdk5 interferes with the microtubule-binding and polymerizing activities of p35. Using a mutational approach, we found that only phosphorylation at Thr-138, one of the two residues primarily phosphorylated in vivo, inhibits the polymerizing activity. In PC12 cells, expression of p35 promotes nerve growth factor-induced neurite outgrowth under a Cdk5 inhibitory condition. Such p35 activity is impaired by the phosphomimetic mutation of Thr-138. These data suggest that Thr-138 phosphorylation plays a critical role in the control of the p35 functions in microtubule assembly and neurite outgrowth.

    The Journal of biological chemistry 2008;283;19;13252-60

  • Cyclin-dependent kinase 5 is associated with risk for Alzheimer's disease in a Dutch population-based study.

    Arias-Vásquez A, Aulchenko YS, Isaacs A, van Oosterhout A, Sleegers K, Hofman A, van Broeckhoven C, Oostra BA, Breteler M and van Duijn CM

    Genetic Epidemiology Unit, Dept. of Epidemiology & Biostatistics, Erasmus Medical Center, PO Box 2040, 3000 CA Rotterdam, The Netherlands. a.ariasvasquez@psy.umcn.nl

    Although the role of the Cdk5 protein in Alzheimer's disease (AD) is well recognized, there have been relatively few studies investigating genetic variants in the CDK5 gene in AD. In this study, we assessed the association between five previously described single nucleotide polymorphisms (SNPs) in the CDK5 gene and late onset AD by means of logistic regression and haplotype association analyses. Including all prevalent and incident AD cases, we found a significantly increased risk of AD for carriers of the GG genotype of SNP rs2069442 (OR = 1.79, 95 % CI 1.16-2.79, p = 0.001) in those without APOE*4. When limiting the analysis to incident cases without APOE*4, carriers of the GG genotype showed a 1.9-fold increased risk of AD (95 % CI 1.16-3.10, p = 0.003). Variations in the CDK5 gene can be described in 5 haplotype blocks. In our analysis, the haplotype tagged by the G allele of SNP rs2069442 was significantly associated with AD (p = 0.05). In conclusion, our study suggests that CDK5 may be associated with AD.

    Journal of neurology 2008;255;5;655-62

  • Enterovirus 71 triggering of neuronal apoptosis through activation of Abl-Cdk5 signalling.

    Chen TC, Lai YK, Yu CK and Juang JL

    Division of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan.

    The molecular mechanism behind what causes an infection of Enterovirus 71 (EV71) in young children to result in severe neurological diseases is unclear. Herein, we show that Cdk5, a critical signalling effector of various neurotoxic insults in the brain, is activated by EV71 infection of neuronal cells. EV71-induced neuronal apoptosis could be effectively repressed by blocking either Cdk5 kinase activity or its protein expression. Moreover, EV71-induced Cdk5 activation was modulated by c-Abl. The suppression of c-Abl kinase activity by STI571 notably repressed both the Cdk5 activation and neuronal apoptosis in cells infected with EV71. Although EV71 also induces apoptosis in non-neuronal cells, it did not affect Abl and Cdk5 activities in several non-neuronal cell lines. Intriguingly, coxsackievirus A16 (CA16), a genetically closely related serotype to EV71 that usually does not induce severe neurological disorders, could only weakly stimulate Abl, but not Cdk5 kinase activity. Taken together, our data suggest a serotype- and cell type-specific mechanism, by which EV71 induces Abl kinase activity, which in turn triggers Cdk5-signalling for neuronal apoptosis.

    Cellular microbiology 2007;9;11;2676-88

  • The activation and inhibition of cyclin-dependent kinase-5 by phosphorylation.

    Zhang B, Tan VB, Lim KM and Tay TE

    Department of Chemistry, Zhejiang University, Hangzhou 310027, PR China.

    Despite the very similar 3-dimensional structures as reflected by the more than 60% identity in amino acid sequences, CDK2 and CDK5 have very different functions and characteristics. Phosphorylation on a conserved Thr14 can inhibit activities of both the kinases, but phosphorylating another conserved Tyr15, however, can lead to totally opposite inhibition and stimulation consequences in CDK2 and CDK5. Our molecular dynamics (MD) simulations suggest a similar inhibition mechanism of phosphorylation on the Thr14 as in the CDK2 system. In both the systems, the kinase activities are inhibited by the phosphorylation because it causes ATP phosphate moiety misalignment and changes in the Mg2+ ion coordination sphere, which have been proven to be critical for the phosphate group of the ATP transferring to the hydroxyl group on the serine in the substrate peptide. The calculations indicate that ATP adopts a more favorable conformation and location in the phosphorylated Tyr15 complex to facilitate the interactions with the substrate and the Mg2+ is wrapped more strongly by the phosphate group than in the unphosphorylated system, which might be favored by the transfer reaction.

    Biochemistry 2007;46;38;10841-51

  • Phosphorylation of huntingtin by cyclin-dependent kinase 5 is induced by DNA damage and regulates wild-type and mutant huntingtin toxicity in neurons.

    Anne SL, Saudou F and Humbert S

    Institut Curie, F-91405 Orsay, France.

    Huntingtin is an antiapoptotic protein that becomes toxic when its polyglutamine stretch is expanded, resulting in Huntington's disease (HD). Protein context and posttranslational modifications regulate huntingtin toxicity. Identifying signaling pathways that act on huntingtin is, therefore, key to understanding huntingtin function in normal and pathological conditions. We show here that huntingtin is phosphorylated by the cyclin-dependent kinase 5 (Cdk5) at serines 1181 and 1201. Phosphorylation can be induced by DNA damage in vitro and in vivo. The state of huntingtin phosphorylation is a crucial regulator of neuronal cell death. Absence of phosphorylation of huntingtin at serines 1181 and 1201 confers toxic properties to wild-type huntingtin in a p53-dependent manner in striatal neurons and accelerates neuronal death induced by DNA damage. In contrast, phosphorylation at serines 1181 and 1201 protects against polyQ-induced toxicity. Finally, we show in late stages of HD a sustained DNA damage that is associated with a decrease in Cdk5/p35 levels. We propose that wild-type huntingtin is a component of the DNA damage response signal in neurons and that the Cdk5/DNA damage pathway is dysregulated in HD.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;27;7318-28

  • Stabilization and activation of p53 induced by Cdk5 contributes to neuronal cell death.

    Lee JH, Kim HS, Lee SJ and Kim KT

    Department of Life Science, Division of Molecular and Life Science, Systems-Biodynamics NCRC, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea.

    The p53 tumor suppressor protein is a key regulator of cellular functions including responses to numerous stress signals, and triggers apoptosis in many cell types, including neurons. The major mechanisms known to regulate p53 stabilization and activation include phosphorylation and ubiquitin ligase-mediated proteasomal degradation. Cyclin-dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase, is most active in the central nervous system and plays a variety of roles in neuronal degeneration. Here, we demonstrate for the first time that Cdk5 interacts with p53 and increases its stability through posttranslational regulation, leading to accumulation of p53, particularly in the nucleus. We show that Cdk5 phosphorylates p53 on Ser15, Ser33 and Ser46 in vitro, and that increased Cdk5 activity in the nucleus mediates these phosphorylation events in response to genotoxic and oxidative stresses. Cdk5 mediates disruption of the interaction between p53 and Hdm2 (also known as Mdm2), and prevents Hdm2-induced p53 ubiquitylation and downregulation. Cdk5 additionally enhances phosphorylation-dependent binding of the p300 coactivator, inducing acetylation of p53. Cdk5-stabilized p53 protein is transcriptionally active, resulting in the induction of pro-apoptotic genes and subsequent mitochondria-mediated apoptosis in response to genotoxic or oxidative stress. Collectively, these novel findings help define the mechanisms underlying neuronal apoptosis occurring as a result of Cdk5-mediated p53 stabilization and transcriptional activation.

    Journal of cell science 2007;120;Pt 13;2259-71

  • Microtubule association of the neuronal p35 activator of Cdk5.

    Hou Z, Li Q, He L, Lim HY, Fu X, Cheung NS, Qi DX and Qi RZ

    Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.

    Cdk5 and its neuronal activator p35 play an important role in neuronal migration and proper development of the brain cortex. We show that p35 binds directly to alpha/beta-tubulin and microtubules. Microtubule polymers but not the alpha/beta-tubulin heterodimer block p35 interaction with Cdk5 and therefore inhibit Cdk5-p35 activity. p25, a neurotoxin-induced and truncated form of p35, does not have tubulin and microtubule binding activities, and Cdk5-p25 is inert to the inhibitory effect of microtubules. p35 displays strong activity in promoting microtubule assembly and inducing formation of microtubule bundles. Furthermore, microtubules stabilized by p35 are resistant to cold-induced disassembly. In cultured cortical neurons, a significant proportion of p35 localizes to microtubules. When microtubules were isolated from rat brain extracts, p35 co-assembled with microtubules, including cold-stable microtubules. Together, these findings suggest that p35 is a microtubule-associated protein that modulates microtubule dynamics. Also, microtubules play an important role in the control of Cdk5 activation.

    The Journal of biological chemistry 2007;282;26;18666-70

  • Phosphorylation of Parkin by the cyclin-dependent kinase 5 at the linker region modulates its ubiquitin-ligase activity and aggregation.

    Avraham E, Rott R, Liani E, Szargel R and Engelender S

    Department of Pharmacology, The B. Rappaport Faculty of Medicine and Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096, Israel.

    Mutations in Parkin are responsible for a large percentage of autosomal recessive juvenile parkinsonism cases. Parkin displays ubiquitin-ligase activity and protects against cell death promoted by several insults. Therefore, regulation of Parkin activities is important for understanding the dopaminergic cell death observed in Parkinson disease. We now report that cyclin-dependent kinase 5 (Cdk5) phosphorylates Parkin both in vitro and in vivo. We found that highly specific Cdk5 inhibitors and a dominant negative Cdk5 construct inhibited Parkin phosphorylation, suggesting that a significant portion of Parkin is phosphorylated by Cdk5. Parkin interacts with Cdk5 as observed by co-immunoprecipitation experiments of transfected cells and rat brains. Phosphorylation by Cdk5 decreased the auto-ubiquitylation of Parkin both in vitro and in vivo. We identified Ser-131 located at the linker region of Parkin as the major Cdk5 phosphorylation site. The Cdk5 phosphorylation-deficient S131A Parkin mutant displayed a higher auto-ubiquitylation level and increased ubiquitylation activity toward its substrates synphilin-1 and p38. Additionally, the S131A Parkin mutant more significantly accumulated into inclusions in human dopaminergic cells when compared with the wild-type Parkin. Furthermore, S131A Parkin mutant increased the formation of synphilin-1/alpha-synuclein inclusions, suggesting that the levels of Parkin phosphorylation and ubiquitylation may modulate the formation of inclusion bodies relevant to the disease. The data indicate that Cdk5 is a new regulator of the Parkin ubiquitin-ligase activity and modulates its ability to accumulate into and modify inclusions. Phosphorylation by Cdk5 may contribute to the accumulation of toxic Parkin substrates and decrease the ability of dopaminergic cells to cope with toxic insults in Parkinson disease.

    The Journal of biological chemistry 2007;282;17;12842-50

  • Role of activation of PIP5Kgamma661 by AP-2 complex in synaptic vesicle endocytosis.

    Nakano-Kobayashi A, Yamazaki M, Unoki T, Hongu T, Murata C, Taguchi R, Katada T, Frohman MA, Yokozeki T and Kanaho Y

    Department of Physiological Chemistry, Graduate School of Comprehensive Human Sciences and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan.

    Synaptic vesicles (SVs) are retrieved by clathrin-mediated endocytosis at the nerve terminals. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] drives this event by recruiting the components of the endocytic machinery. However, the molecular mechanisms that result in local generation of PI(4,5)P2 remain unclear. We demonstrate here that AP-2 complex directly interacts with phosphatidylinositol 4-phosphate 5-kinase gamma661 (PIP5Kgamma661), the major PI(4,5)P2-producing enzyme in the brain. The beta2 subunit of AP-2 was found to bind to the C-terminal tail of PIP5Kgamma661 and cause PIP5Kgamma661 activation. The interaction is regulated by PIP5Kgamma661 dephosphorylation, which is triggered by depolarization in mouse hippocampal neurons. Finally, overexpression of the PIP5Kgamma661 C-terminal region in hippocampal neurons suppresses depolarization-dependent SV endocytosis. These findings provide evidence for the molecular mechanism through which PIP5Kgamma661 locally generates PI(4,5)P2 in hippocampal neurons and suggest a model in which the interaction trigger SV endocytosis.

    Funded by: NIGMS NIH HHS: R01 GM071520, R01 GM71520

    The EMBO journal 2007;26;4;1105-16

  • Cdk5 regulates STAT3 activation and cell proliferation in medullary thyroid carcinoma cells.

    Lin H, Chen MC, Chiu CY, Song YM and Lin SY

    Department of Life Science, National Chung Hsing University, Taichung 40227, Taiwan. hlin@dragon.nchu.edu.tw

    The biological behaviors of thyroid cancer are varied, and the pathological mechanisms remain unclear. Some reports indicated an apparent aggregation of amyloid accompanying medullary thyroid carcinoma (MTC). Amyloid aggregation in neurodegeneration leads to hyperactivation of Cdk5 and subsequent neuronal death. Based on the connection with amyloid, the role of Cdk5 in MTC is worthy of investigation. Initially, the expression of Cdk5 and its activator, p35, in MTC cell lines was identified. Cdk5 inhibition by specific inhibitors or short interfering RNA decreased the proliferation of MTC cell lines, which reveals the importance of Cdk5 in MTC cell growth. Although p35 cleavage has been considered as an important element in neurodegeneration, it seems that p35 cleavage was not a major cause in Cdk5 activity-dependent MTC cell proliferation because neither Cdk5 activity nor cell growth was affected by the inhibition of p35 cleavage. Clearance of amyloid by antibody neutralization indicated that MTC cell proliferation was supported by calcitonin-derived extracellular amyloid and subsequent Her2 and Cdk5 activation. Significantly, the STAT3 pathway was involved in Cdk5-dependent proliferation of MTC cells through Ser-727 phosphorylation. In addition, Cdk5 inhibition reduced nuclear distributions of both the Cdk5-p35 complex and phospho-STAT3 in MTC cells. Finally, Cdk5 inhibition retarded tumor formation in vivo accompanying the reduction of phospho-STAT3. Our findings suggest the first demonstration of a novel and specific role for Cdk5 kinase in supporting the proliferation of the medullary thyroid carcinoma cells and could shed light on a new field for diagnosis and therapy of thyroid cancer.

    The Journal of biological chemistry 2007;282;5;2776-84

  • Suppression of calpain-dependent cleavage of the CDK5 activator p35 to p25 by site-specific phosphorylation.

    Kamei H, Saito T, Ozawa M, Fujita Y, Asada A, Bibb JA, Saido TC, Sorimachi H and Hisanaga S

    Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachiohji, Tokyo 192-0397, Japan.

    Cdk5 is a proline-directed Ser/Thr protein kinase predominantly expressed in postmitotic neurons together with its activator, p35. N-terminal truncation of p35 to p25 by calpain results in deregulation of Cdk5 and contributes to neuronal cell death associated with several neurodegenerative diseases. Previously we reported that p35 occurred as a phosphoprotein, phospho-p35 levels changed with neuronal maturation, and that phosphorylation of p35 affected its vulnerability to calpain cleavage. Here, we identify the p35 residues Ser(8) and Thr(138) as the major sites of phosphorylation by Cdk5. Mutagenesis of these sites to unphosphorylatable Ala increased susceptibility to calpain in cultured cells and neurons while changing them to phosphomimetic glutamate-attenuated cleavage. Furthermore, phosphorylation state-specific antibodies to these sites revealed that Thr(138) was dephosphorylated in adult rat, although both Ser(8) and Thr(138) were phosphorylated in prenatal brains. In cultured neurons, inhibition of protein phosphatases converted phosho-Ser(8) p35 to dual phospho-Ser(8)/Thr(138) p35 and conferred resistance to calpain cleavage. These results suggest phosphorylation of Thr(138) predominantly defines the susceptibility of p35 to calpain-dependent cleavage and that dephosphorylation of this site is a critical determinant of Cdk5-p25-induced cell death associated with neurodegeneration.

    The Journal of biological chemistry 2007;282;3;1687-94

  • Expression of cyclin-dependent kinase 5 mRNA and protein in the human brain following acute ischemic stroke.

    Mitsios N, Pennucci R, Krupinski J, Sanfeliu C, Gaffney J, Kumar P, Kumar S, Juan-Babot O and Slevin M

    School of Biology, Chemistry and Health Science, Manchester Metropolitan University, Manchester, UK.

    Neuronal cell death after brain ischemia may be regulated by activation of cyclin-dependent kinase 5 (Cdk5). In this study, expression of Cdk5 and its activator p35/p25 was examined in human post-mortem stroke tissue and in human cerebral cortical fetal neurons and human brain microvascular endothelial cells exposed to oxygen-glucose deficiency and reperfusion. The majority of patients demonstrated increased expression of Cdk5 and p-Cdk5 in stroke-affected tissue, with about a third showing increased p35 and p25 cleaved fragment as determined by Western blotting. An increase in Cdk5-, p-Cdk5- and p35-positive neurons and microvessels occurred in stroke-affected regions of patients. Staining of neurons became irregular and clumped in the cytoplasm, and nuclear translocation occurred, with colocalization of p35 and Cdk5. Association of Cdk5 with nuclear damage was demonstrated by coexpression of nuclear Cdk5 in TUNEL-positive neurons and microvessels in peri-infarcted regions. In vitro studies showed up-regulation and/or nuclear translocation of Cdk5, p-Cdk5 and p35 in neurons and endothelial cells subjected to oxygen-glucose deficiency, and strong staining was associated with propidium iodide positive nuclei, an indicator of cellular damage. These results provide new evidence for a role of Cdk5 in the events associated with response to ischemic injury in humans.

    Brain pathology (Zurich, Switzerland) 2007;17;1;11-23

  • Identification of nuclear import mechanisms for the neuronal Cdk5 activator.

    Fu X, Choi YK, Qu D, Yu Y, Cheung NS and Qi RZ

    Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.

    The activation of Cdk5 by p35 plays a pivotal role in a multitude of nervous system activities ranging from neuronal differentiation to degeneration. A fraction of Cdk5 and p35 localizes in the nucleus where Cdk5-p35 exerts its functions via protein phosphorylation, and p35 displays a dynamic localization between the cytoplasm and the nucleus. Here, we examined the nuclear import properties of p35. In nuclear import assays, p35 was actively transported into the nuclei of digitonin-permeabilized HeLa cells and cortical neurons by cytoplasmic carrier-mediated mechanisms. Importin-beta, importin-5, and importin-7 were identified to import p35 into the nuclei via a direct interaction with it. An N-terminal region of p35 was defined to interact with the above importins, serving as a nuclear localization signal. Finally, we show that the nuclear localization of p35 does not require the association of Cdk5. Furthermore, Cdk5 and importin-beta/5/7 are mutually exclusive in binding to p35. These results suggest that p35 employs pathways distinct from that used by Cdk5 for transport to the nucleus.

    The Journal of biological chemistry 2006;281;51;39014-21

  • PKA modulates GSK-3beta- and cdk5-catalyzed phosphorylation of tau in site- and kinase-specific manners.

    Liu F, Liang Z, Shi J, Yin D, El-Akkad E, Grundke-Iqbal I, Iqbal K and Gong CX

    Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA. feiliu63@hotmail.com

    Phosphorylation of tau protein is regulated by several kinases, especially glycogen synthase kinase 3beta (GSK-3beta), cyclin-dependent protein kinase 5 (cdk5) and cAMP-dependent protein kinase (PKA). Phosphorylation of tau by PKA primes it for phosphorylation by GSK-3beta, but the site-specific modulation of GSK-3beta-catalyzed tau phosphorylation by the prephosphorylation has not been well investigated. Here, we found that prephosphorylation by PKA promotes GSK-3beta-catalyzed tau phosphorylation at Thr181, Ser199, Ser202, Thr205, Thr217, Thr231, Ser396 and Ser422, but inhibits its phosphorylation at Thr212 and Ser404. In contrast, the prephosphorylation had no significant effect on its subsequent phosphorylation by cdk5 at Thr181, Ser199, Thr205, Thr231 and Ser422; inhibited it at Ser202, Thr212, Thr217 and Ser404; and slightly promoted it at Ser396. These studies reveal the nature of the inter-regulation of tau phosphorylation by the three major tau kinases.

    Funded by: NIA NIH HHS: AG016760, AG019158, AG027429, R01 AG016760, R01 AG019158, R01 AG027429

    FEBS letters 2006;580;26;6269-74

  • Regulation of phosphorylation of tau by cyclin-dependent kinase 5 and glycogen synthase kinase-3 at substrate level.

    Sengupta A, Novak M, Grundke-Iqbal I and Iqbal K

    Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314-6399, USA.

    Microtubule associated protein tau, which is expressed in six alternatively spliced molecular isoforms in human brain, is abnormally hyperphosphorylated in Alzheimer disease and related tauopathies. Here, we show (i) that GSK-3alpha and neither GSK-3beta nor cdk5 can phosphorylate tau at Ser262 and phosphorylation at Ser235 by cdk5 primes phosphorylation at Thr231 by GSK-3alpha/beta; (ii) that tau isoforms with two N-terminal inserts (tau4L, tau3L) are phosphorylated by cdk5 plus GSK-3 at Thr231 markedly more than isoforms lacking these inserts (tau4, tau3); and (iii) that Thr231 is phosphorylated approximately 50% more in free tau than in microtubule-bound tau, and the phosphorylation at this site results in the dissociation of tau from microtubules. These findings suggest that the phosphorylation of tau at Thr231 and Ser262 by cdk5 plus GSK-3, which inhibits its normal biological activity, is regulated both by its amino terminal inserts and its physical state.

    Funded by: NIA NIH HHS: AG019158, AG05892, AG08776, R01 AG019158, R01 AG019158-08; NINDS NIH HHS: NS18105

    FEBS letters 2006;580;25;5925-33

  • A nestin scaffold links Cdk5/p35 signaling to oxidant-induced cell death.

    Sahlgren CM, Pallari HM, He T, Chou YH, Goldman RD and Eriksson JE

    Turku Centre for Biotechnology, University of Turku and Abo Akademi University, Turku, Finlandand.

    The intermediate filament protein, nestin, has been implicated as an organizer of survival-determining signaling molecules. When nestin expression was related to the sensitivity of neural progenitor cells to oxidant-induced apoptosis, nestin displayed a distinct cytoprotective effect. Oxidative stress in neuronal precursor cells led to downregulation of nestin with subsequent activation of cyclin-dependent kinase 5 (Cdk5), a crucial kinase in the nervous system. Nestin downregulation was a prerequisite for the Cdk5-dependent apoptosis, as overexpression of nestin efficiently inhibited induction of apoptosis, whereas depletion of nestin by RNA interference had a sensitizing effect. When the underlying link between nestin and Cdk5 was analyzed, we observed that nestin serves as a scaffold for Cdk5, with binding restricted to a specific region following the alpha-helical domain of nestin, and that the presence and organization of nestin regulated the sequestration and activity of Cdk5, as well as the ubiquitylation and turnover of its regulator, p35. Our data imply that nestin is a survival determinant whose action is based upon a novel mode of Cdk5 regulation, affecting the targeting, activity, and turnover of the Cdk5/p35 signaling complex.

    The EMBO journal 2006;25;20;4808-19

  • Phosphorylation of WAVE1 regulates actin polymerization and dendritic spine morphology.

    Kim Y, Sung JY, Ceglia I, Lee KW, Ahn JH, Halford JM, Kim AM, Kwak SP, Park JB, Ho Ryu S, Schenck A, Bardoni B, Scott JD, Nairn AC and Greengard P

    Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.

    WAVE1--the Wiskott-Aldrich syndrome protein (WASP)--family verprolin homologous protein 1--is a key regulator of actin-dependent morphological processes in mammals, through its ability to activate the actin-related protein (Arp2/3) complex. Here we show that WAVE1 is phosphorylated at multiple sites by cyclin-dependent kinase 5 (Cdk5) both in vitro and in intact mouse neurons. Phosphorylation of WAVE1 by Cdk5 inhibits its ability to regulate Arp2/3 complex-dependent actin polymerization. Loss of WAVE1 function in vivo or in cultured neurons results in a decrease in mature dendritic spines. Expression of a dephosphorylation-mimic mutant of WAVE1 reverses this loss of WAVE1 function in spine morphology, but expression of a phosphorylation-mimic mutant does not. Cyclic AMP (cAMP) signalling reduces phosphorylation of the Cdk5 sites in WAVE1, and increases spine density in a WAVE1-dependent manner. Our data suggest that phosphorylation/dephosphorylation of WAVE1 in neurons has an important role in the formation of the filamentous actin cytoskeleton, and thus in the regulation of dendritic spine morphology.

    Funded by: NIDA NIH HHS: P01 DA010044

    Nature 2006;442;7104;814-7

  • Different mechanisms of CDK5 and CDK2 activation as revealed by CDK5/p25 and CDK2/cyclin A dynamics.

    Otyepka M, Bártová I, Kríz Z and Koca J

    Department of Physical Chemistry and Center for Biomolecules and Complex Molecular Systems, Palacký University, tr. Svobody 26, 771 46 Olomouc, Czech Republic. otyepka@aix.upol.cz

    A detailed analysis is presented of the dynamics of human CDK5 in complexes with the protein activator p25 and the purine-like inhibitor roscovitine. These and other findings related to the activation of CDK5 are critically reviewed from a molecular perspective. In addition, the results obtained on the behavior of CDK5 are compared with data on CDK2 to assess the differences and similarities between the two kinases in terms of (i) roscovitine binding, (ii) regulatory subunit association, (iii) conformational changes in the T-loop following CDK/regulatory subunit complex formation, and (iv) specificity in CDK/regulatory subunit recognition. An energy decomposition analysis, used for these purposes, revealed why the binding of p25 alone is sufficient to stabilize the extended active T-loop conformation of CDK5, whereas the equivalent conformational change in CDK2 requires both the binding of cyclin A and phosphorylation of the Thr(160) residue. The interaction energy of the CDK5 T-loop with p25 is about 26 kcal.mol(-1) greater than that of the CDK2 T-loop with cyclin A. The binding pattern between CDK5 and p25 was compared with that of CDK2/cyclin A to find specific regions involved in CDK/regulatory subunit recognition. The analyses performed revealed that the alphaNT-helix of cyclin A interacts with the alpha6-alpha7 loop and the alpha7 helix of CDK2, but these regions do not interact in the CDK5/p25 complex. Further differences between the CDK5/p25 and CDK2/cyclin A systems studied are discussed with respect to their specific functionality.

    The Journal of biological chemistry 2006;281;11;7271-81

  • Cyclin-dependent protein kinase 5 primes microtubule-associated protein tau site-specifically for glycogen synthase kinase 3beta.

    Li T, Hawkes C, Qureshi HY, Kar S and Paudel HK

    Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec H3T 1E2, Canada.

    In the preceding paper, we showed that GSK3beta phosphorylates tau at S(202), T(231), S(396), and S(400) in vivo. Phosphorylation of S(202) occurs without priming. Phosphorylation of T(231), on the other hand, requires priming phosphorylation of S(235). Similarly, priming phosphorylation of S(404) is essential for the sequential phosphorylation of S(400) and S(396) by GSK3beta. The priming kinase that phosphorylates tau at S(235) and S(404) in the brain is not known. In this study, we find that in HEK-293 cells cotransfected with tau, GSK3beta, and Cdk5, Cdk5 phosphorylates tau at S(202), S(235), and S(404). S(235) phosphorylation enhances GSK3beta-catalyzed T(231) phosphorylation. Similarly, Cdk5 by phosphorylating S(404) stimulates phosphorylation of S(400) and S(396) by GSK3beta. These data indicate that Cdk5 primes tau for GSK3beta in intact cells. To evaluate if Cdk5 primes tau for GSK3beta in mammalian brain, we examined localizations of Cdk5, tau, and GSK3beta in rat brain. We also analyzed the interaction of Cdk5 with tau and GSK3beta in brain microtubules. We found that Cdk5, GSK3beta, and tau are virtually colocalized in rat brain cortex. When bovine brain microtubules are analyzed by FPLC gel filtration, Cdk5, GSK3beta, and tau coelute within an approximately 450 kDa complex. From the fractions containing the approximately 450 kDa complex, tau, Cdk5, and GSK3beta co-immunoprecipitate with each other. In HEK-293 cells transfected with tau, Cdk5, and GSK3beta in different combinations, tau binds to Cdk5 in a manner independent of GSK3beta and to GSK3beta in a manner independent of Cdk5. However, Cdk5 and GSK3beta bind to each other only in the presence of tau, suggesting that tau connects Cdk5 and GSK3beta. Our results suggest that in the brain, tau, Cdk5, and GSK3beta are components of an approximately 450 kDa complex. Within the complex, Cdk5 phosphorylates tau at S(235) and primes it for phosphorylation of T(231) by GSK3beta. Similarly, Cdk5 by phosphorylating tau at S(404) primes tau for a sequential phosphorylation of S(400) and S(396) by GSK3beta.

    Biochemistry 2006;45;10;3134-45

  • Cyclin-dependent kinase 5, Munc18a and Munc18-interacting protein 1/X11alpha protein up-regulation in Alzheimer's disease.

    Jacobs EH, Williams RJ and Francis PT

    Wolfson Centre for Age-Related Diseases, Guy's, King's and St Thomas' Schools of Biomedical Sciences, King's College London, St. Thomas Street, London SE1 1UL, UK. e.jacobs@erasmusmc.nl

    Besides formation of neurofibrillary tangles and neuron loss, the Alzheimer's disease brain is characterized by neuritic plaques consisting of beta-amyloid peptide deposits and impaired neurotransmission. The proteins Munc18a, Munc18-interacting protein 1 and Munc18-interacting protein 2 mediate exocytosis and decrease beta-amyloid peptide formation. Cyclin-dependent kinase 5 and its activator p35 disrupt Munc18a-syntaxin 1 binding, thereby promoting synaptic vesicle fusion during exocytosis. We investigated protein levels of the signaling pathway: p35, cyclin-dependent kinase 5, Munc18a, syntaxin 1A and 1B, Munc18-interacting protein 1 and Munc18-interacting protein 2 in Alzheimer's disease cortex and found that this pathway was up-regulated in the Alzheimer's disease parietal and occipital cortex. In the cortex of transgenic Tg2576 mice over-expressing human beta-amyloid precursor protein with the Swedish mutation known to lead to familial Alzheimer's disease, which have substantial levels of beta-amyloid peptide but lack neurofibrillary tangles and neuron loss, no alterations of protein levels were detected. These data suggest that the pathway is enhanced in dying or surviving neurons and might serve a protective role by compensating for decreased neurotransmission and decreasing beta-amyloid peptide levels early during the progression of Alzheimer's disease.

    Neuroscience 2006;138;2;511-22

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • Association of cyclin-dependent kinase 5 and neuronal activators p35 and p39 complex in early-onset Alzheimer's disease.

    Rademakers R, Sleegers K, Theuns J, Van den Broeck M, Bel Kacem S, Nilsson LG, Adolfsson R, van Duijn CM, Van Broeckhoven C and Cruts M

    Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB8), University of Antwerp, Belgium.

    Malfunctioning of cyclin-dependent kinase 5 (CDK5) through aberrant proteolytic cleavage of its neuronal activators p35 and p39 is involved in neurodegeneration in Alzheimer's disease (AD) and other neurodegenerative brain diseases. By extensive genetic analysis of the genes encoding CDK5 (CDK5), p35 (CDK5R1) and p39 (CDK5R2), we excluded causal mutations in 70 familial early-onset AD patients. We performed an association study with five informative SNPs in CDK5 in two independent samples of early-onset AD patients and matched control individuals from The Netherlands and northern Sweden. Association was observed with g.149800G>C in intron 5 of CDK5, and a two times increased risk was observed in both patient samples for carriers of the C-allele. Our data are indicative for a role of the CDK5 molecular complex in the genetic etiology of early-onset AD, and suggest that a yet unknown functional variant in CDK5 or in a nearby gene might lead to increased susceptibility for early-onset AD.

    Neurobiology of aging 2005;26;8;1145-51

  • Molecular basis for the specificity of p27 toward cyclin-dependent kinases that regulate cell division.

    Lacy ER, Wang Y, Post J, Nourse A, Webb W, Mapelli M, Musacchio A, Siuzdak G and Kriwacki RW

    Department of Structural Biology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.

    The cyclin-dependent kinase inhibitors (CKIs) bind to and directly regulate the catalytic activity of cyclin-dependent kinase (Cdk)/cyclin complexes involved in cell cycle control and do not regulate other, closely related Cdks. We showed previously that the CKI, p27, binds to Cdk2/cyclin A though a sequential mechanism that involves folding-on-binding. The first step in the kinetic mechanism is interaction of a small, highly dynamic domain of p27 (domain 1) with the cyclin subunit of the Cdk2/cyclin A complex, followed by much slower binding of a more lengthy and less flexible domain (domain 2) to Cdk2. The second step requires folding of domain 2 into the kinase inhibitory conformation. Rapid binding of p27 domain 1 to cyclin A tethers the inhibitor to the binary Cdk2/cyclin A complex, which reduces the entropic barrier associated with slow binding of domain 2 to the catalytic subunit. We show here that p27/cyclin interactions are an important determinant of p27 specificity towards cell cycle Cdks. We used surface plasmon resonance, limited proteolysis, mass spectrometry, and NMR spectroscopy to study the interaction of p27 with Cdk2/cyclin A, and with another Cdk complex, Cdk5/p25, that is involved in neurodegeneration. Importantly, Cdk5/p35 (the parent complex of Cdk5/p25) is not regulated by p27 in neurons. Our results show that p27 binds to Cdk5 and Cdk2 with similar, slow kinetics. However, p27 fails to interact with p25 within the Cdk5/p25 complex, which we believe prevents formation of a kinetically trapped, inhibited p27/Cdk5/p25 complex in vivo. The helical topology of p25 is very similar to that of cyclin A. However, p25 lacks the MRAIL sequence in one helix that, in the cell cycle cyclins, mediates specific interactions with domain 1 of p21 and p27. Our results strongly suggest that p21 and p27, related Cdk inhibitors, select their cell cycle regulatory Cdk targets by binding specifically to the cyclin subunit of these Cdk/cyclin complexes as a first step in a sequential, folding-on-binding mechanism.

    Funded by: NCI NIH HHS: P30 CA 21765, R01 CA 82491

    Journal of molecular biology 2005;349;4;764-73

  • Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases: implications for mutant huntingtin toxicity.

    Luo S, Vacher C, Davies JE and Rubinsztein DC

    Department of Medical Genetics, Cambridge Institute for Medical Research, Addenbrooke's Hospital, Cambridge, CB2 2XY, England, UK.

    Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) tract in the huntingtin (htt) protein. Mutant htt toxicity is exposed after htt cleavage by caspases and other proteases release NH(2)-terminal fragments containing the polyQ expansion. Here, we show htt interacts and colocalizes with cdk5 in cellular membrane fractions. Cdk5 phosphorylates htt at Ser434, and this phosphorylation reduces caspase-mediated htt cleavage at residue 513. Reduced mutant htt cleavage resulting from cdk5 phosphorylation attenuated aggregate formation and toxicity in cells expressing the NH(2)-terminal 588 amino acids (htt588) of mutant htt. Cdk5 activity is reduced in the brains of HD transgenic mice compared with controls. This result can be accounted for by the polyQ-expanded htt fragments reducing the interaction between cdk5 and its activator p35. These data predict that the ability of cdk5 phosphorylation to protect against htt cleavage, aggregation, and toxicity is compromised in cells expressing toxic fragments of htt.

    Funded by: Medical Research Council: G0000872; Wellcome Trust

    The Journal of cell biology 2005;169;4;647-56

  • Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution.

    Cheng J, Kapranov P, Drenkow J, Dike S, Brubaker S, Patel S, Long J, Stern D, Tammana H, Helt G, Sementchenko V, Piccolboni A, Bekiranov S, Bailey DK, Ganesh M, Ghosh S, Bell I, Gerhard DS and Gingeras TR

    Affymetrix Inc., Santa Clara, CA 95051, USA.

    Sites of transcription of polyadenylated and nonpolyadenylated RNAs for 10 human chromosomes were mapped at 5-base pair resolution in eight cell lines. Unannotated, nonpolyadenylated transcripts comprise the major proportion of the transcriptional output of the human genome. Of all transcribed sequences, 19.4, 43.7, and 36.9% were observed to be polyadenylated, nonpolyadenylated, and bimorphic, respectively. Half of all transcribed sequences are found only in the nucleus and for the most part are unannotated. Overall, the transcribed portions of the human genome are predominantly composed of interlaced networks of both poly A+ and poly A- annotated transcripts and unannotated transcripts of unknown function. This organization has important implications for interpreting genotype-phenotype associations, regulation of gene expression, and the definition of a gene.

    Science (New York, N.Y.) 2005;308;5725;1149-54

  • p25/Cdk5-mediated retinoblastoma phosphorylation is an early event in neuronal cell death.

    Hamdane M, Bretteville A, Sambo AV, Schindowski K, Bégard S, Delacourte A, Bertrand P and Buée L

    INSERM U422, Institut de Médecine Prédictive et Recherche Thérapeutique, Université de Lille 2, Place de Verdun, 59045 Lille Cedex, France.

    In large models of neuronal cell death, there is a tight correlation between Cdk5 deregulation and cell-cycle dysfunction. However, pathways that link Cdk5 to the cell cycle during neuronal death are still unclear. We have investigated the molecular events that precede p25/Cdk5-triggered neuronal death using a neuronal cell line that allows inducible p25 expression. In this system, no sign of apoptosis was seen before 24 hours of p25 induction. Thus, at that time, cell-cycle-regulatory proteins were analysed by immunoblotting and some of them showed a significant deregulation. Interestingly, after time-course experiments, the earliest feature correlated with p25 expression was the phosphorylation of the retinoblastoma protein (Rb). Indeed, this phosphorylation was observed 6 hours after p25 induction and was abolished in the presence of a Cdk5 inhibitor, roscovitine, which does not inhibit the usual Rb cyclin-D kinases Cdk4 and Cdk6. Furthermore, analyses of levels and subcellular localization of Cdk-related cyclins did not reveal any change following Cdk5 activation, arguing for a direct effect of Cdk5 activity on Rb protein. This latter result was clearly demonstrated by in vitro kinase assays showing that the p25-Cdk5 complex in our cell system phosphorylates Rb directly without the need for any intermediary kinase activity. Hence, Rb might be an appropriate candidate that connects Cdk5 to cell-cycle deregulation during neuronal cell death.

    Journal of cell science 2005;118;Pt 6;1291-8

  • GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity.

    Yoshimura T, Kawano Y, Arimura N, Kawabata S, Kikuchi A and Kaibuchi K

    Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa-ku, Nagoya, Aichi 466-8550, Japan.

    Neurons are highly polarized and comprised of two structurally and functionally distinct parts, an axon and dendrites. We previously showed that collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate, possibly by promoting neurite elongation via microtubule assembly. Here, we showed that glycogen synthase kinase-3beta (GSK-3beta) phosphorylated CRMP-2 at Thr-514 and inactivated it. The expression of the nonphosphorylated form of CRMP-2 or inhibition of GSK-3beta induced the formation of multiple axon-like neurites in hippocampal neurons. The expression of constitutively active GSK-3beta impaired neuronal polarization, whereas the nonphosphorylated form of CRMP-2 counteracted the inhibitory effects of GSK-3beta, indicating that GSK-3beta regulates neuronal polarity through the phosphorylation of CRMP-2. Treatment of hippocampal neurons with neurotrophin-3 (NT-3) induced inactivation of GSK-3beta and dephosphorylation of CRMP-2. Knockdown of CRMP-2 inhibited NT-3-induced axon outgrowth. These results suggest that NT-3 decreases phosphorylated CRMP-2 and increases nonphosphorylated active CRMP-2, thereby promoting axon outgrowth.

    Cell 2005;120;1;137-49

  • GTP-dependent secretion from neutrophils is regulated by Cdk5.

    Rosales JL, Ernst JD, Hallows J and Lee KY

    Department of Cell Biology and Anatomy, Cancer Biology and Neuroscience Research Groups, The University of Calgary, Calgary, Alberta T2N 4N1, Canada. rosales@ucalgary.ca

    We have previously shown evidence for the existence of a calcium-independent, GTP-regulated mechanism of secretion from neutrophils, but this secretory mechanism remains to be fully elucidated. Cyclin-dependent kinase 5 (Cdk5), the various substrates of which include Munc18 and synapsin 1, has been implicated in neuronal secretion. Although the Cdk5 activator, p35, and Cdk5-p35 activity are primarily associated with neurons, we report here that p35 also exists in neutrophils and that an active Cdk5-p35 complex is present in these cells. Cdk5-p35 activity in human neutrophils is mostly localized in secretory granules, which show an increase in Cdk5-p35 level and activity upon GTP stimulation. The potent Cdk5 inhibitor, roscovitine, completely blocks GTP-stimulated granule Cdk5 activity, which accompanies lactoferrin secretion from neutrophil-specific granules. Roscovitine also inhibits GTP-induced lactoferrin secretion and surface localization of the secretion markers, CD63 and CD66b, to a certain extent. Furthermore, neutrophils from wild-type mice treated with roscovitine and neutrophils from p35(-/-) mice exhibit comparable surface expression levels of both CD63 and CD66b upon GTP stimulation. Although our data suggest that other molecules control GTP-induced secretion from neutrophils, it is clear that Cdk5-p35 is required to elicit the maximum GTP-induced secretory response. Our observation that multiple proteins in neutrophil granules serve as specific substrates of Cdk5 further supports the premise that the kinase is a key component of the GTP-regulated secretory apparatus in neutrophils.

    The Journal of biological chemistry 2004;279;52;53932-6

  • Phosphoproteomic analysis of the developing mouse brain.

    Ballif BA, Villén J, Beausoleil SA, Schwartz D and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Proper development of the mammalian brain requires the precise integration of numerous temporally and spatially regulated stimuli. Many of these signals transduce their cues via the reversible phosphorylation of downstream effector molecules. Neuronal stimuli acting in concert have the potential of generating enormous arrays of regulatory phosphoproteins. Toward the global profiling of phosphoproteins in the developing brain, we report here the use of a mass spectrometry-based methodology permitting the first proteomic-scale phosphorylation site analysis of primary animal tissue, identifying over 500 protein phosphorylation sites in the developing mouse brain.

    Funded by: NHGRI NIH HHS: HG00041

    Molecular & cellular proteomics : MCP 2004;3;11;1093-101

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Sequence comparison of human and mouse genes reveals a homologous block structure in the promoter regions.

    Suzuki Y, Yamashita R, Shirota M, Sakakibara Y, Chiba J, Mizushima-Sugano J, Nakai K and Sugano S

    Human Genome Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, 108-8639, Japan. ysuzuki@ims.u-tokyo.ac.jp

    Comparative sequence analysis was carried out for the regions adjacent to experimentally validated transcriptional start sites (TSSs), using 3324 pairs of human and mouse genes. We aligned the upstream putative promoter sequences over the 1-kb proximal regions and found that the sequence conservation could not be further extended at, on average, 510 bp upstream positions of the TSSs. This discontinuous manner of the sequence conservation revealed a "block" structure in about one-third of the putative promoter regions. Consistently, we also observed that G+C content and CpG frequency were significantly different inside and outside the blocks. Within the blocks, the sequence identity was uniformly 65% regardless of their length. About 90% of the previously characterized transcription factor binding sites were located within those blocks. In 46% of the blocks, the 5' ends were bounded by interspersed repetitive elements, some of which may have nucleated the genomic rearrangements. The length of the blocks was shortest in the promoters of genes encoding transcription factors and of genes whose expression patterns are brain specific, which suggests that the evolutional diversifications in the transcriptional modulations should be the most marked in these populations of genes.

    Genome research 2004;14;9;1711-8

  • Involvement of Cdk5/p25 in digoxin-triggered prostate cancer cell apoptosis.

    Lin H, Juang JL and Wang PS

    Division of Molecular and Genomic Medicine, National Health Research Institutes, Taipei 115, Taiwan, Republic of China.

    Cardiac digitalis has been considered to be a treatment for breast cancer. Our previous study indicates that digoxin, one member in digitalis, decreases the proliferation of prostate cancer cells, but the mechanisms remain unclear. In the present study, Ca(2+) proved to be an important factor in digoxin-triggered prostate cancer cell death. Because cyclin-dependent kinase (Cdk)5 and p35 cleavage (p25 formation) have been reported to be targets of intracellular Ca(2+), and subsequently correlated to apoptosis, we not only demonstrated first that Cdk5, p35, and p25 proteins were all expressed in prostate cancer cells (including lymph node carcinoma of the prostate (LNCaP) and DU-145 cells), but also showed where p25 formation and Cdk5 kinase activity were affected by treatment with digoxin. The inhibitor of p35 cleavage (calpeptin) was used to reduce p25 formation, and the result suggested that p25 accumulation might be the major cause of digoxin-triggered LNCaP cell death. Butyrolactone-I and roscovitine, two Cdk5 kinase inhibitors, were also found to prevent digoxin-triggered LNCaP cell death. In addition, treatment of siRNA-Cdk5 diminished digoxin-triggered cell death, as compared with the treatments of siRNA-Cdk1 or siRNA-Cdk2, which implies the specific involvement of Cdk5 in digoxin-triggered cell death. Caspase inhibitor set and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay were used to demonstrate that digoxin-triggered LNCaP cell apoptosis through Cdk5 activation. These results suggest that Cdk5/p35 and p25 are novel players in digoxin-triggered prostate cancer cell apoptosis and, therefore, become potential therapeutic targets.

    The Journal of biological chemistry 2004;279;28;29302-7

  • The T-cell protein tyrosine phosphatase is phosphorylated on Ser-304 by cyclin-dependent protein kinases in mitosis.

    Bukczynska P, Klingler-Hoffmann M, Mitchelhill KI, Lam MH, Ciccomancini M, Tonks NK, Sarcevic B, Kemp BE and Tiganis T

    Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3800, Australia.

    Two alternatively spliced forms of the human protein tyrosine phosphatase TCPTP (T-cell protein tyrosine phosphatase) exist: a 48 kDa form that is targeted to the endoplasmic reticulum (TC48) and a shorter 45 kDa form that is targeted to the nucleus (TC45). In this study we have identified Ser-304 (Phe301-Asp-His-Ser304-Pro-Asn-Lys307) as a major TCPTP phosphory-lation site and demonstrate that TC45, but not TC48, is phosphorylated on this site in vivo. Phosphorylation of TC45 on Ser-304 was cell cycle-dependent, and increased as cells progressed from G2 into mitosis, but subsided upon mitotic exit. Ser-304 phosphorylation was increased when cells were arrested in mitosis by microtubule poisons such as nocodazole, but remained unaltered when cells were arrested at the G2/M checkpoint by adriamycin. Phosphorylation of Ser-304 did not alter significantly the phosphatase activity or the protein stability of TC45, and had no apparent effect on TC45 localization. Ser-304 phosphorylation was ablated when cells were treated with the CDK (cyclin-dependent protein kinase) inhibitors roscovitine or SU9516, but remained unaltered when ERK1/2 activation was inhibited with the MEK (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase) inhibitor PD98059. In addition, recombinant CDKs, but not the Polo-like kinase Plk1, phosphorylated Ser-304 in vitro. Our studies identify Ser-304 as a major phosphorylation site in human TCPTP, and the TC45 variant as a novel mitotic CDK substrate.

    Funded by: NCI NIH HHS: CA53840

    The Biochemical journal 2004;380;Pt 3;939-49

  • Suppression of the caspase cleavage of beta-amyloid precursor protein by its cytoplasmic phosphorylation.

    Taru H, Yoshikawa K and Suzuki T

    Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi -6, Kita-ku, Sapporo 060-0812, Japan.

    beta-Amyloid precursor protein (APP) is a type I transmembrane protein. Its cleavages by beta- and gamma-secretases yield beta-amyloid, which is the main constituent of senile plaques in Alzheimer's disease (AD). In apoptotic cells and AD brains, APP is alternatively cleaved by caspases in the cytoplasmic region after the Asp664 residue (with respect to the numbering conversion for the APP695 isoform). Caspase-cleaved fragments of APP are cytotoxic and have been implicated in AD pathogenesis; however, the mechanisms regulating the cleavage have not been studied. APP is constitutively phosphorylated at Thr668 in brain. In the present study, we demonstrate that APP phosphorylated at Thr668 is less vulnerable to cytoplasmic cleavage by caspase-3 and caspase-8. This suggests that APP phosphorylation suppresses the generation of caspase-cleaved fragments of APP in the brain and that perturbation of this phosphorylation may be involved in APP-mediated neurotoxicity.

    FEBS letters 2004;567;2-3;248-52

  • Alpha-chimaerin exists in a functional complex with the Cdk5 kinase in brain.

    Qi RZ, Ching YP, Kung HF and Wang JH

    Institute of Molecular Biology, University of Hong Kong, Pokfulam, Hong Kong.

    Cyclin-dependent kinase 5 (Cdk5) in association with its neuronal activators p35 and p39 shows a complex involvement in the control of neurocytoskeletal dynamics. Here we show that alpha-chimaerin, a GTPase-activating protein specific for Rac and Cdc42, is a p35-binding protein. The interaction domains of p35 and alpha-chimaerin were delineated. In transfected HeLa cells, p35 and alpha-chimaerin displayed an overlapping distribution pattern and they could be co-immunoprecipitated from the cell lysate. As alpha-chimaerin has a regulatory function in actin repolymerization, these results suggested that the regulation of neurocytoskeleton dynamics by Cdk5 is mediated at least in part via alpha-chimaerin.

    FEBS letters 2004;561;1-3;177-80

  • Cdk5 phosphorylation of doublecortin ser297 regulates its effect on neuronal migration.

    Tanaka T, Serneo FF, Tseng HC, Kulkarni AB, Tsai LH and Gleeson JG

    Department of Neurosciences, University of California, San Diego, La Jolla, 92093, USA.

    Mutations in the doublecortin (DCX) gene in human or targeted disruption of the cdk5 gene in mouse lead to similar cortical lamination defects in the developing brain. Here we show that Dcx is phosphorylated by Cdk5. Dcx phosphorylation is developmentally regulated and corresponds to the timing of expression of p35, the major activating subunit for Cdk5. Mass spectrometry and Western blot analysis indicate phosphorylation at Dcx residue Ser297. Phosphorylation of Dcx lowers its affinity to microtubules in vitro, reduces its effect on polymerization, and displaces it from microtubules in cultured neurons. Mutation of Ser297 blocks the effect of Dcx on migration in a fashion similar to pharmacological inhibition of Cdk5 activity. These results suggest that Dcx phosphorylation by Cdk5 regulates its actions on migration through an effect on microtubules.

    Funded by: NINDS NIH HHS: R01 NS041537; PHS HHS: N5047101

    Neuron 2004;41;2;215-27

  • Regulation of synaptojanin 1 by cyclin-dependent kinase 5 at synapses.

    Lee SY, Wenk MR, Kim Y, Nairn AC and De Camilli P

    Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06510, USA.

    Synaptojanin 1 is a polyphosphoinositide phosphatase concentrated in presynaptic nerve terminals, where it dephosphorylates a pool of phosphatidylinositol 4,5-bisphosphate implicated in synaptic vesicle recycling. Like other proteins with a role in endocytosis, synaptojanin 1 undergoes constitutive phosphorylation in resting synapses and stimulation-dependent dephosphorylation by calcineurin. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates synaptojanin 1 and regulates its function both in vitro and in intact synaptosomes. Cdk5 phosphorylation inhibited the inositol 5-phosphatase activity of synaptojanin 1, whereas dephosphorylation by calcineurin stimulated such activity. The activity of synaptojanin 1 was also stimulated by its interaction with endophilin 1, its major binding partner at the synapse. Notably, Cdk5 phosphorylated serine 1144, which is adjacent to the endophilin binding site. Mutation of serine 1144 to aspartic acid to mimic phosphorylation by Cdk5 inhibited the interaction of synaptojanin 1 with endophilin 1. These results suggest that Cdk5 and calcineurin may have an antagonistic role in the regulation of synaptojanin 1 recruitment and activity, and therefore in the regulation of phosphatidylinositol 4,5-bisphosphate turnover at synapses.

    Funded by: NCI NIH HHS: CA46128, P01 CA046128; NIDA NIH HHS: DA10044, P01 DA010044; NINDS NIH HHS: NS36251, R01 NS036251, R37 NS036251

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;2;546-51

  • Co-purification and localization of Munc18-1 (p67) and Cdk5 with neuronal cytoskeletal proteins.

    Bhaskar K, Shareef MM, Sharma VM, Shetty AP, Ramamohan Y, Pant HC, Raju TR and Shetty KT

    Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore 560029, India.

    Munc18-1 (p67, nSec1, rbSec1), a neuron-specific 67kDa protein was independently identified as a syntaxin-binding protein, and as a component that co-purifies with, and regulates the kinase activity of cyclin dependent kinase (Cdk5). Gene knockout studies have demonstrated a role for Munc18-1 in synaptic vesicle docking and neurotransmitter release. Mice lacking Munc18-1 gene were synaptically silent, but the gene deletion did not prevent normal brain assembly, including the formation of layered structures, fiber pathways and morphologically defined synapses. Previous study has shown that Munc18-1 facilitates Cdk5 mediated phosphorylation of KSPXK domains of the neuronal cytoskeletal elements, suggesting that Munc18-1 may function in the regulation of cytoskeletal dynamics. Present study demonstrates the co-purification and co-localization of Munc18 with cytoskeletal elements and forms first step towards understanding the role for Munc18-1 in cytoskeletal dynamics. Conversely, the cytoskeletal proteins and Cdk5 co-purifies with Munc18-1 in a Munc18-1 immuno-affinity chromatography, suggesting a strong protein-protein interaction. Findings from immunofluorescence studies in PC12 cells have shown co-localization of Munc18-1 and Cdk5 with neurofilaments and microtubules. Further, immunohistochemical and immuno-electron microscopic studies of rat olfactory bulb also demonstrated co-localization of Munc18-1 and Cdk5 with cytoskeletal elements. Thus, the biochemical evidence of strong interaction between Munc18-1 with cytoskeletal proteins and morphological evidence of their (Munc18 and cytoskeletal elements) identical sub-cellular localization is suggestive of the possible role for Munc18-1 in cytoskeletal dynamics.

    Neurochemistry international 2004;44;1;35-44

  • Apoptosis-associated tyrosine kinase is a Cdk5 activator p35 binding protein.

    Honma N, Asada A, Takeshita S, Enomoto M, Yamakawa E, Tsutsumi K, Saito T, Satoh T, Itoh H, Kaziro Y, Kishimoto T and Hisanaga S

    Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Hachiohji, Tokyo 192-0397, Japan.

    A 3(')-terminal fragment of a splice variant of KIAA0641, a human homologue of apoptosis-associated tyrosine kinase (AATYK), was screened from human brain cDNA libraries by a yeast two-hybrid system using a Cdk5 activator p35 as a bait. The cloned cDNA encoded 477 amino acids, composed of internal 458 amino acids of KIAA0641 and 19 amino acids unique to this variant after splicing, then referred to this clone as hAATYKs-p35BP (human AATYK short isoform-p35 binding polypeptide). Using GST-fusion protein, hAATYKs-p35BP was shown to bind to Cdk5/p35 in a rat brain extract. hAATYKs made by fusing the kinase domain of KIAA0641 to the N-terminus of hAATYKs-p35BP was used for binding to Cdk5/p35 in HEK293 cells. Both hAATYKs and KIAA0641 bound to and were phosphorylated by Cdk5/p35. These results suggest that both isoforms of hAATYK are novel Cdk5/p35-binding and substrate proteins.

    Biochemical and biophysical research communications 2003;310;2;398-404

  • Puralpha is essential for postnatal brain development and developmentally coupled cellular proliferation as revealed by genetic inactivation in the mouse.

    Khalili K, Del Valle L, Muralidharan V, Gault WJ, Darbinian N, Otte J, Meier E, Johnson EM, Daniel DC, Kinoshita Y, Amini S and Gordon J

    Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania 19122, USA. kamel.khalili@temple.edu

    The single-stranded DNA- and RNA-binding protein, Puralpha, has been implicated in many biological processes, including control of transcription of multiple genes, initiation of DNA replication, and RNA transport and translation. Deletions of the PURA gene are frequent in acute myeloid leukemia. Mice with targeted disruption of the PURA gene in both alleles appear normal at birth, but at 2 weeks of age, they develop neurological problems manifest by severe tremor and spontaneous seizures and they die by 4 weeks. There are severely lower numbers of neurons in regions of the hippocampus and cerebellum of PURA(-/-) mice versus those of age-matched +/+ littermates, and lamination of these regions is aberrant at time of death. Immunohistochemical analysis of MCM7, a protein marker for DNA replication, reveals a lack of proliferation of precursor cells in these regions in the PURA(-/-) mice. Levels of proliferation were also absent or low in several other tissues of the PURA(-/-) mice, including those of myeloid lineage, whereas those of PURA(+/-) mice were intermediate. Evaluation of brain sections indicates a reduction in myelin and glial fibrillary acidic protein labeling in oligodendrocytes and astrocytes, respectively, indicating pathological development of these cells. At postnatal day 5, a critical time for cerebellar development, Puralpha and Cdk5 were both at peak levels in bodies and dendrites of Purkinje cells of PURA(+/+) mice, but both were absent in dendrites of PURA(-/-) mice. Puralpha and Cdk5 can be coimmunoprecipitated from brain lysates of PURA(+/+) mice. Immunohistochemical studies reveal a dramatic reduction in the level of both phosphorylated and nonphosphorylated neurofilaments in dendrites of the Purkinje cell layer and of synapse formation in the hippocampus. Overall results are consistent with a role for Puralpha in developmentally timed DNA replication in specific cell types and also point to a newly emerging role in compartmentalized RNA transport and translation in neuronal dendrites.

    Molecular and cellular biology 2003;23;19;6857-75

  • Cyclin-dependent kinase-5 is involved in neuregulin-dependent activation of phosphatidylinositol 3-kinase and Akt activity mediating neuronal survival.

    Li BS, Ma W, Jaffe H, Zheng Y, Takahashi S, Zhang L, Kulkarni AB and Pant HC

    Laboratory of Neurochemistry, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20892-4130, USA.

    The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays an important role in mediating survival signals in wide variety of neurons and cells. Recent studies show that Akt also regulates metabolic pathways to regulate cell survival. In this study, we reported that cyclin-dependent kinase-5 (Cdk5) regulates Akt activity and cell survival through the neuregulin-mediated PI 3-kinase signaling pathway. We found that brain extracts of Cdk5-/-mice display a lower PI 3-kinase activity and phosphorylation of Akt compared with that in wild type mice. Moreover, we demonstrated that Cdk5 phosphorylated Ser-1176 in the neuregulin receptor ErbB2 and phosphorylated Thr-871 and Ser-1120 in the ErbB3 receptor. We identified the Ser-1120 sequence RSRSPR in ErbB3 as a novel phosphorylation consensus sequence of Cdk5. Finally, we found that Cdk5 activity is involved in neuregulin-induced Akt activity and neuregulin-mediated neuronal survival. These findings suggest that Cdk5 may exert a key role in promoting neuronal survival by regulating Akt activity through the neuregulin/PI 3-kinase signaling pathway.

    The Journal of biological chemistry 2003;278;37;35702-9

  • Mitotic-like tau phosphorylation by p25-Cdk5 kinase complex.

    Hamdane M, Sambo AV, Delobel P, Bégard S, Violleau A, Delacourte A, Bertrand P, Benavides J and Buée L

    INSERM U422, IMPRT, Place de Verdun, 59045 Lille, France and CNS Research, Aventis Pharma, 94400 Vitry Sur Seine, France.

    Among tau phosphorylation sites, some phosphoepitopes referred to as abnormal ones are exclusively found on tau aggregated into filaments in Alzheimer's disease. Recent data suggested that molecular mechanisms similar to those encountered during mitosis may play a role in abnormal tau phosphorylation. In particular, TG-3 phosphoepitope is associated with early stages of neurofibrillary tangles (NFTs). In this study, we reported a suitable cell model consisting of SH-SY5Y cells stably transfected with an inducible p25 expression vector. It allows investigation of tau phosphorylation by p25-Cdk5 kinase complex in a neuronal context and avoiding p25-induced cytotoxicity. Immunoblotting analyses showed that p25-Cdk5 strongly phosphorylates tau protein not only at the AT8 epitope but also at the AT180 epitope and at the Alzheimer's mitotic epitope TG-3. Further biochemical analyses showed that abnormal phosphorylated tau accumulated in cytosol as a microtubule-free form, suggesting its impact on tau biological activity. Since tau abnormal phosphorylation occurred in dividing cells, TG-3 immunoreactivity was also investigated in differentiated neuronal ones, and both TG-3-immunoreactive tau and nucleolin, another early marker for NFT, were also generated. These data suggest that p25-Cdk5 is responsible for the mitotic-like phosphoepitopes present in NFT and argue for a critical role of Cdk5 in neurodegenerative mechanisms.

    The Journal of biological chemistry 2003;278;36;34026-34

  • Cdk5 is essential for synaptic vesicle endocytosis.

    Tan TC, Valova VA, Malladi CS, Graham ME, Berven LA, Jupp OJ, Hansra G, McClure SJ, Sarcevic B, Boadle RA, Larsen MR, Cousin MA and Robinson PJ

    Cell Signalling Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145, Australia.

    Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained c4 by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin I on Ser 774 and Ser 778 in vitr 1f40 o, which are identical to its endogenous phosphorylation sites in vivo. Cdk5 antagonists and expression of dominant-negative Cdk5 block phosphorylation of dynamin I, but not of amphiphysin or AP180, in nerve terminals and inhibit SVE. Thus Cdk5 has an essential role in SVE and is the first dephosphin kinase identified in nerve terminals.

    Nature cell biology 2003;5;8;701-10

  • 14-3-3epsilon is important for neuronal migration by binding to NUDEL: a molecular explanation for Miller-Dieker syndrome.

    Toyo-oka K, Shionoya A, Gambello MJ, Cardoso C, Leventer R, Ward HL, Ayala R, Tsai LH, Dobyns W, Ledbetter D, Hirotsune S and Wynshaw-Boris A

    Department of Pediatrics, UCSD Cancer Center, University of California, San Diego School of Medicine, 9500 Gilman Drive, Mailstop 0627, La Jolla, California 92093-0627, USA.

    Heterozygous deletions of 17p13.3 result in the human neuronal migration disorders isolated lissencephaly sequence (ILS) and the more severe Miller-Dieker syndrome (MDS). Mutations in PAFAH1B1 (the gene encoding LIS1) are responsible for ILS and contribute to MDS, but the genetic causes of the greater severity of MDS are unknown. Here, we show that the gene encoding 14-3-3epsilon (YWHAE), one of a family of ubiquitous phosphoserine/threonine-binding proteins, is always deleted in individuals with MDS. Mice deficient in Ywhae have defects in brain development and neuronal migration, similar to defects observed in mice heterozygous with respect to Pafah1b1. Mice heterozygous with respect to both genes have more severe migration defects than single heterozygotes. 14-3-3epsilon binds to CDK5/p35-phosphorylated NUDEL and this binding maintains NUDEL phosphorylation. Similar to LIS1, deficiency of 14-3-3epsilon results in mislocalization of NUDEL and LIS1, consistent with reduction of cytoplasmic dynein function. These results establish a crucial role for 14-3-3epsilon in neuronal development by sustaining the effects of CDK5 phosphorylation and provide a molecular explanation for the differences in severity of human neuronal migration defects with 17p13.3 deletions.

    Nature genetics 2003;34;3;274-85

  • Cdk5 regulates the organization of Nestin and its association with p35.

    Sahlgren CM, Mikhailov A, Vaittinen S, Pallari HM, Kalimo H, Pant HC and Eriksson JE

    Department of Biology, Abo Akademi University, BioCity, Finland.

    The intermediate filament protein nestin is characterized by its specific expression during the development of neuronal and myogenic tissues. We identify nestin as a novel in vivo target for cdk5 and p35 kinase, a critical signaling determinant in development. Two cdk5-specific phosphorylation sites on nestin, Thr-1495 and Thr-316, were established, the latter of which was used as a marker for cdk5-specific phosphorylation in vivo. Ectopic expression of cdk5 and p35 in central nervous system progenitor cells and in myogenic precursor cells induced elevated phosphorylation and reorganization of nestin. The kinetics of nestin expression corresponded to elevated expression and activation of cdk5 during differentiation of myoblast cell cultures and during regeneration of skeletal muscle. In the myoblasts, a disassembly-linked phosphorylation of Thr-316 indicated active phosphorylation of nestin by cdk5. Moreover, cdk5 occurred in physical association with nestin. Inhibition of cdk5 activity-either by transfection with dominant-negative cdk5 or by using a specific cdk5 inhibitor-blocked myoblast differentiation and phosphorylation of nestin at Thr-316, and this inhibition markedly disturbed the organization of nestin. Interestingly, the interaction between p35, the cdk5 activator, and nestin appeared to be regulated by cdk5. In differentiating myoblasts, p35 was not complexed with nestin phosphorylated at Thr-316, and inhibition of cdk5 activity during differentiation induced a marked association of p35 with nestin. These results demonstrate that there is a continuous turnover of cdk5 and p35 activity on a scaffold formed by nestin. This association is likely to affect the organization and operation of both cdk5 and nestin during development.

    Molecular and cellular biology 2003;23;14;5090-106

  • Identification of a novel, membrane-associated neuronal kinase, cyclin-dependent kinase 5/p35-regulated kinase.

    Kesavapany S, Lau KF, Ackerley S, Banner SJ, Shemilt SJ, Cooper JD, Leigh PN, Shaw CE, McLoughlin DM and Miller CC

    Departments of Neuroscience and Neurology, The Institute of Psychiatry, Kings College London, London SE5 8AF, United Kingdom.

    Here we characterize a novel neuronal kinase, cyclin-dependent kinase 5 (cdk5)/p35-regulated kinase (cprk). Cprk is a member of a previously undescribed family of kinases that are predicted to contain two N-terminal membrane-spanning domains and a long C terminus, which harbors a dual-specificity serine/threonine/tyrosine kinase domain. Cprk was isolated in a yeast two-hybrid screen using the neuronal cdk5 activator p35 as "bait." Cprk interacts with p35 in the yeast-two hybrid system, binds to p35 in glutathione S-transferase fusion pull-down assays, and colocalizes with p35 in cultured neurons and transfected cells. In these cells, cprk is present with p35 in the Golgi apparatus. Cprk is expressed in a number of tissues but is enriched in brain and muscle and within the brain is found in a wide range of neuronal populations. Cprk displays catalytic activity in in vitro kinase assays and is itself phosphorylated by cdk5/p35. Cdk5/p35 inhibits cprk activity. Cdk5/p35 may therefore regulate cprk function in the brain.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2003;23;12;4975-83

  • Cdk5 is a key factor in tau aggregation and tangle formation in vivo.

    Noble W, Olm V, Takata K, Casey E, Mary O, Meyerson J, Gaynor K, LaFrancois J, Wang L, Kondo T, Davies P, Burns M, Veeranna, Nixon R, Dickson D, Matsuoka Y, Ahlijanian M, Lau LF and Duff K

    Center for Dementia Research, Nathan S. Kline Institute, New York University, 140 Old Orangeburg Road, Orangeburg, NY 10962, USA.

    Tau aggregation is a common feature of neurodegenerative diseases such as Alzheimer's disease, and hyperphosphorylation of tau has been implicated as a fundamental pathogenic mechanism in this process. To examine the impact of cdk5 in tau aggregation and tangle formation, we crossed transgenic mice overexpressing the cdk5 activator p25, with transgenic mice overexpressing mutant (P301L) human tau. Tau was hyperphosphorylated at several sites in the double transgenics, and there was a highly significant accumulation of aggregated tau in brainstem and cortex. This was accompanied by increased numbers of silver-stained neurofibrillary tangles (NFTs). Insoluble tau was also associated with active GSK. Thus, cdk5 can initiate a major impact on tau pathology progression that probably involves several kinases. Kinase inhibitors may thus be beneficial therapeutically.

    Funded by: NIA NIH HHS: AG 172116

    Neuron 2003;38;4;555-65

  • Human chromosome 7: DNA sequence and biology.

    Scherer SW, Cheung J, MacDonald JR, Osborne LR, Nakabayashi K, Herbrick JA, Carson AR, Parker-Katiraee L, Skaug J, Khaja R, Zhang J, Hudek AK, Li M, Haddad M, Duggan GE, Fernandez BA, Kanematsu E, Gentles S, Christopoulos CC, Choufani S, Kwasnicka D, Zheng XH, Lai Z, Nusskern D, Zhang Q, Gu Z, Lu F, Zeesman S, Nowaczyk MJ, Teshima I, Chitayat D, Shuman C, Weksberg R, Zackai EH, Grebe TA, Cox SR, Kirkpatrick SJ, Rahman N, Friedman JM, Heng HH, Pelicci PG, Lo-Coco F, Belloni E, Shaffer LG, Pober B, Morton CC, Gusella JF, Bruns GA, Korf BR, Quade BJ, Ligon AH, Ferguson H, Higgins AW, Leach NT, Herrick SR, Lemyre E, Farra CG, Kim HG, Summers AM, Gripp KW, Roberts W, Szatmari P, Winsor EJ, Grzeschik KH, Teebi A, Minassian BA, Kere J, Armengol L, Pujana MA, Estivill X, Wilson MD, Koop BF, Tosi S, Moore GE, Boright AP, Zlotorynski E, Kerem B, Kroisel PM, Petek E, Oscier DG, Mould SJ, Döhner H, Döhner K, Rommens JM, Vincent JB, Venter JC, Li PW, Mural RJ, Adams MD and Tsui LC

    Department of Genetics and Genomic Biology, The Hospital for Sick Children, Toronto, Ontario, Canada, M5G 1X8. steve@genet.sickkids.on.ca

    DNA sequence and annotation of the entire human chromosome 7, encompassing nearly 158 million nucleotides of DNA and 1917 gene structures, are presented. To generate a higher order description, additional structural features such as imprinted genes, fragile sites, and segmental duplications were integrated at the level of the DNA sequence with medical genetic data, including 440 chromosome rearrangement breakpoints associated with disease. This approach enabled the discovery of candidate genes for developmental diseases including autism.

    Funded by: Canadian Institutes of Health Research: 38103; NIGMS NIH HHS: P01 GM061354

    Science (New York, N.Y.) 2003;300;5620;767-72

  • Increased ezrin expression and activation by CDK5 coincident with acquisition of the senescent phenotype.

    Yang HS and Hinds PW

    Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.

    Passage of normal cells in culture leads to senescence, an irreversible cell cycle exit characterized by biochemical changes and a distinctive morphology. Cellular stresses, including oncogene activation, can also lead to senescence. Consistent with an anti-oncogenic role for this process, the tumor suppressor pRb plays a critical role in senescence. Reexpression of pRb in human tumor cells results in senescence-like changes including cell cycle exit and shape changes. Here we show that senescence is accompanied by increased expression and altered localization of ezrin, an actin binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of CDK5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells.

    Funded by: NIA NIH HHS: AG20208

    Molecular cell 2003;11;5

  • Cdk5-mediated inhibition of the protective effects of transcription factor MEF2 in neurotoxicity-induced apoptosis.

    Gong X, Tang X, Wiedmann M, Wang X, Peng J, Zheng D, Blair LA, Marshall J and Mao Z

    The Liver Research Center, Department of Medicine, Rhode Island Hospital, Brown University School of Medicine, Providence, Rhode Island 02903, USA.

    Neurotoxic insults deregulate Cdk5 activity, which leads to neuronal apoptosis and may contribute to neurodegeneration. The biological activity of Cdk5 has been ascribed to its phosphorylation of cytoplasmic substrates. However, its roles in the nucleus remain unknown. Here we investigate the mechanism by which Cdk5 promotes neuronal apoptosis. We have identified the prosurvival transcription factor MEF2 as a direct nuclear target of Cdk5. Cdk5 phosphorylates MEF2 at a distinct serine in its transactivation domain to inhibit MEF2 activity. Neurotoxicity enhances nuclear Cdk5 activity, leading to Cdk5-dependent phosphorylation and inhibition of MEF2 function in neurons. MEF2 mutants resistant to Cdk5 phosphorylation restore MEF2 activity and protect primary neurons from Cdk5 and neurotoxin-induced apoptosis. Our studies reveal a nuclear pathway by which neurotoxin/Cdk5 induces neuronal apoptosis through inhibiting prosurvival nuclear machinery.

    Funded by: NICHD NIH HHS: HD39446-01; NINDS NIH HHS: R01 NS37676-01A1, R01 NS39063-01A2

    Neuron 2003;38;1;33-46

  • Tat-induced deregulation of neuronal differentiation and survival by nerve growth factor pathway.

    Peruzzi F, Gordon J, Darbinian N and Amini S

    Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania 19122, USA.

    HIV-1 enters the brain at the early stage of infection and resides primarily in a limited number of macrophages/microglia and astrocytes. Infection of these cells, however, may not explain the massive neuronal pathology which is seen in AIDS-associated dementia, suggesting a role for factors released from HIV-1 infected cells that trigger a cascade of events leading to neurodegeneration. Our results indicate that Tat, the potent regulatory protein of HIV-1 which is secreted by infected cells and can affect neighboring uninfected cells by transcellular means, can influence multiple biological events that lead to neuronal injury. These findings demonstrate that treatment of neuronal cells with Tat affects MAPK/ERK1/2 activity, the downstream central component of the nerve growth factor (NGF) signaling pathway. Furthermore, our data indicate that treatment of cells with Tat severely decreases expression of p35, a neuron-specific activator of cdk5, a cyclin dependent kinase that phosphorylates several neuronal proteins including neurofilament, and plays an important role in neuronal differentiation and survival. In parallel, Tat can bind to the cellular protein, Puralpha, which associates with cdk5. Further, results from Puralpha knockout animals revealed a decrease in p35 activity, pointing to the importance of Puralpha association with cdk5 in the activity of cdk5:p35 complex. These data demonstrate the cooperativity between HIV-1 Tat and the Puralpha in deregulation of the NGF signal transduction pathway in neuronal cells.

    Journal of neurovirology 2002;8 Suppl 2;91-6

  • Truncation of CDK5 activator p35 induces intensive phosphorylation of Ser202/Thr205 of human tau.

    Hashiguchi M, Saito T, Hisanaga S and Hashiguchi T

    Department of Physiology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku, Japan. Mhashigu@aol.com

    Hyperphosphorylated tau is a major component of neurofibrillary tangles, one of the hallmarks of Alzheimer's disease. CDK5 is a kinase that phosphorylates the tau protein, and its endogenous activator, p35, regulates its activity. Recently, calpain was found to digest p35 to its truncated product, p25. Several lines of evidence suggest that p25-CDK5 has much more powerful kinase activity and that it may cause abnormal hyperphosphorylation of tau. In this study, we have examined the kinetic characteristics of in vitro phosphorylation of the longest isoform of human tau by CDK5 and its activators using recombinant proteins. Although the kinase activity of CDK5 in phosphorylating tau was significantly higher in the presence of p25, the affinity of CDK5 for tau was not different. Phosphopeptide mapping revealed enhanced phosphorylation of Ser(202)/Thr(205) residues by p25-CDK5 (amino acid residues of tau are numbered according to the longest isoform of human tau). These results suggest that cleavage of p35 to p25 greatly enhances the kinase activity of CDK5 and increases the phosphorylation of Ser(202)/Thr(205). Considering the fact that phosphorylation of Ser(202)/Thr(205) antagonizes the tau-mediated nucleation of tubulin, p25-CDK5 may play a pivotal role in neuronal cell death in Alzheimer's disease.

    The Journal of biological chemistry 2002;277;46;44525-30

  • Involvement of aberrant glycosylation in phosphorylation of tau by cdk5 and GSK-3beta.

    Liu F, Iqbal K, Grundke-Iqbal I and Gong CX

    Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island 10314, USA.

    Microtubule-associated protein tau is abnormally hyperphosphorylated, glycosylated, and aggregated in affected neurons in the brains of individuals with Alzheimer's disease (AD). We recently found that the glycosylation might precede hyperphosphorylation of tau in AD. In this study, we investigated the effect of glycosylation on phosphorylation of tau catalyzed by cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase-3beta (GSK-3beta). The phosphorylation of the longest isoform of recombinant human brain tau, tau(441), at various sites was detected by Western blots and by radioimmuno-dot-blot assay with phosphorylation-dependent and site-specific tau antibodies. We found that cdk5 phosphorylated tau(441) at Thr-181, Ser-199, Ser-202, Thr-205, Thr-212, Ser-214, Thr-217, Thr-231, Ser-235, Ser-396, and Ser-404, but not at Ser-262, Ser-400, Thr-403, Ser-409, Ser-413, or Ser-422. GSK-3beta phosphorylated all the cdk5-catalyzed sites above except Ser-235. Deglycosylation by glycosidases depressed the subsequent phosphorylation of AD-tau (i) with cdk5 at Thr-181, Ser-199, Ser-202, Thr-205, and Ser-404, but not at Thr-212; and (ii) with GSK-3beta at Thr-181, Ser-202, Thr-205, Ser-217, and Ser-404, but not at Ser-199, Thr-212, Thr-231, or Ser-396. These data suggest that aberrant glycosylation of tau in AD might be involved in neurofibrillary degeneration by promoting abnormal hyperphosphorylation by cdk5 and GSK-3beta.

    Funded by: NIA NIH HHS: AG16760, AG19158; NINDS NIH HHS: MN/NS 31862

    FEBS letters 2002;530;1-3;209-14

  • Molecular model of cyclin-dependent kinase 5 complexed with roscovitine.

    Filgueira de Azevedo W, Gaspar RT, Canduri F, Camera JC and Freitas da Silveira NJ

    Departamento de Fi;sica, IBILCE, UNESP 15054-000, São José do Rio Preto, SP, Brazil. walterfa@df.ibile.unesp.br

    Here is described a structural model for the binary complex CDK5-roscovitine. Roscovitine has been shown to potently inhibit cyclin-dependent kinases 1, 2 and 5 (CDK1, 2, and 5), and the structure of CDK2 complexed with roscovitine has been reported; however, no structural data are available for complexes of CDK5 with inhibitors. The structural model indicates that roscovitine strongly binds to the ATP-binding pocket of CDK5 and structural comparison of the CDK2-roscovitine complex correlates the structural differences with differences in inhibition of these CDKs by this inhibitor. This structure opens the possibility of testing new inhibitor families, in addition to new substituents for the already known lead structures of adenine derivatives.

    Biochemical and biophysical research communications 2002;297;5;1154-8

  • The cyclin-dependent kinase 5 activators p35 and p39 interact with the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II and alpha-actinin-1 in a calcium-dependent manner.

    Dhavan R, Greer PL, Morabito MA, Orlando LR and Tsai LH

    Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Cyclin-dependent kinase 5 (Cdk5) is a critical regulator of neuronal migration in the developing CNS, and recent studies have revealed a role for Cdk5 in synaptogenesis and regulation of synaptic transmission. Deregulation of Cdk5 has been linked to the pathology of neurodegenerative diseases such as Alzheimer's disease. Activation of Cdk5 requires its association with a regulatory subunit, and two Cdk5 activators, p35 and p39, have been identified. To gain further insight into the functions of Cdk5, we identified proteins that interact with p39 in a yeast two-hybrid screen. In this study we report that alpha-actinin-1 and the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKIIalpha), two proteins localized at the postsynaptic density, interact with Cdk5 via their association with p35 and p39. CaMKIIalpha and alpha-actinin-1 bind to distinct regions of p35 and p39 and also can interact with each other. The association of CaMKIIalpha and alpha-actinin-1 to the Cdk5 activators, as well as to each other, is stimulated by calcium. Further, the activation of glutamate receptors increases the association of p35 and p39 with CaMKIIalpha, and the inhibition of CaMKII activation diminishes this effect. The glutamate-mediated increase in association of p35 and CaMKIIalpha is mediated in large part by NMDA receptors, suggesting that cross talk between the Cdk5 and CaMKII signal transduction pathways may be a component of the complex molecular mechanisms contributing to synaptic plasticity, memory, and learning.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;18;7879-91

  • A novel centrosome-associated protein with affinity for microtubules.

    Stein PA, Toret CP, Salic AN, Rolls MM and Rapoport TA

    Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115-6091, USA.

    We have identified a novel mammalian protein, MIR1, with microtubule-binding activity. MIR1 is a relative of MID1/midin, the protein implicated in Opitz G/BBB syndrome. In tissue culture cells, MIR1 is enriched at the centrosome. MIR1 dissociates from centrosomes at the G2/M transition and is recruited back to spindle poles during anaphase. When overexpressed during interphase, MIR1 binds along microtubule filaments, which become stabilized, bundled and detached from the centrosome. In mitosis, overexpressed MIR1 dissociates from microtubules but still affects the normally focused localization of gamma-tubulin in spindle poles. Tight binding to microtubules in interphase appears to require an oligomeric state of MIR1, and phosphorylation in mitosis at predicted cyclin-dependent kinase (cdk) sites weakens the interaction.

    Journal of cell science 2002;115;Pt 17;3389-402

  • Pctaire1 interacts with p35 and is a novel substrate for Cdk5/p35.

    Cheng K, Li Z, Fu WY, Wang JH, Fu AK and Ip NY

    Department of Biochemistry, Biotechnology Research Institute and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.

    Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that plays important roles during central nervous system development. Cdk5 kinase activity depends on its regulatory partners, p35 or p39, which are prominently expressed in the central nervous system. We have previously demonstrated the involvement of Cdk5 in the regulation of acetylcholine receptor expression at the neuromuscular junction, suggesting a novel functional role of Cdk5 at the synapse. Here we report the identification of Pctaire1, a member of the Cdk-related kinase family, as a p35-interacting protein in muscle. Binding of Pctaire1 to p35 can be demonstrated by in vitro binding assay and co-immunoprecipitation experiments. Pctaire1 is associated with p35 in cultured myotubes and skeletal muscle, and is concentrated at the neuromuscular junction. Furthermore, Pctaire1 can be phosphorylated by the Cdk5/p25 complex, and serine 95 is the major phosphorylation site. In brain and muscle of Cdk5 null mice, Pctaire1 activity is significantly reduced. Moreover, Pctaire1 activity is increased following preincubation with brain extracts and phosphorylation by the Cdk5/p25 complex. Taken together, our findings demonstrate that Pctaire1 interacts with p35, both in vitro and in vivo, and that phosphorylation of Pctaire1 by Cdk5 enhances its kinase activity.

    The Journal of biological chemistry 2002;277;35;31988-93

  • Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex.

    Sasaki Y, Cheng C, Uchida Y, Nakajima O, Ohshima T, Yagi T, Taniguchi M, Nakayama T, Kishida R, Kudo Y, Ohno S, Nakamura F and Goshima Y

    Department of Molecular Pharmacology and Neurobiology, Yokohama City University School of Medicine, Yokohama, Japan. yukio.sasaki@pharmac.med.yokohama-cu.ac.jp

    Semaphorin-3A (Sema3A), a member of class 3 semaphorins, regulates axon and dendrite guidance in the nervous system. How Sema3A and its receptors plexin-As and neuropilins regulate neuronal guidance is unknown. We observed that in fyn- and cdk5-deficient mice, Sema3A-induced growth cone collapse responses were attenuated compared to their heterologous controls. Cdk5 is associated with plexin-A2 through the active state of Fyn. Sema3A promotes Cdk5 activity through phosphorylation of Tyr15, a phosphorylation site with Fyn. A Cdk5 mutant (Tyr15 to Ala) shows a dominant-negative effect on the Sema3A-induced collapse response. The sema3A gene shows strong interaction with fyn for apical dendrite guidance in the cerebral cortex. We propose a signal transduction pathway in which Fyn and Cdk5 mediate neuronal guidance regulated by Sema3A.

    Neuron 2002;35;5;907-20

  • Cyclin-dependent kinase 5 phosphorylates disabled 1 independently of Reelin signaling.

    Keshvara L, Magdaleno S, Benhayon D and Curran T

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

    Two major signaling pathways that control neuronal positioning during brain development have been uncovered as a result of genetic and biochemical studies on neurological mouse mutants. Mice deficient in Reelin, Disabled 1 (Dab1), or both the very low-density lipoprotein receptor (VLDLR) and the apolipoprotein E receptor 2 (ApoER2) exhibit identical neuroanatomic defects in laminar structures throughout the brain. These proteins function as components of the Reelin signaling pathway. Reelin is a secreted glycoprotein that binds to VLDLR and ApoER2, inducing tyrosine phosphorylation of Dab1, an intracellular adapter protein. Neuronal migration is also regulated by cyclin-dependent kinase 5 (Cdk5) and its activating subunits p35 and p39. Mice deficient in Cdk5, p35, or both p35 and p39 exhibit lamination defects that are similar but not identical to those observed in mice with a defect in the Reelin signaling pathway. Cdk5 phosphorylates proteins that maintain cytoskeletal structures and promote cell motility. To explore the possibility that Cdk5 influences the Reelin pathway, we sought to determine whether Dab1 is a substrate for Cdk5. Here we show that Cdk5 phosphorylates Dab1 on serine 491 in vitro and in vivo, independently of Reelin signaling. We also show that ectopic neurons in Cdk5-deficient mice exhibit reduced levels of Reelin signaling during later stages of cortical development, although Cdk5 is not required for Reelin-induced tyrosine phosphorylation of Dab1. Although the functional significance of Dab1 serine phosphorylation is unclear, our results suggest that there is biochemical cross-talk between two signaling pathways that control cell positioning.

    Funded by: NCI NIH HHS: P30 CA21765; NINDS NIH HHS: R01-NS36558

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;12;4869-77

  • Colocalization and fluorescence resonance energy transfer between cdk5 and AT8 suggests a close association in pre-neurofibrillary tangles and neurofibrillary tangles.

    Augustinack JC, Sanders JL, Tsai LH and Hyman BT

    Department of Neurology, Harvard Medical School, Massachusetts General Hospital, Charlestown 02129, USA.

    Cyclin-dependent kinase 5 (cdk5) is a serine/threonine kinase that, when activated, induces neurite outgrowth. Recent in vitro studies have shown that cdk5 phosphorylates tau at serine 199, serine 202, and threonine 205 and that p25, an activator of cdk5, is increased in Alzheimer disease (AD). Since tau is hyperphosphorylated at these sites in neurofibrillary tangles, we examined brain tissue from patients with AD and normal elderly control cases to determine whether cdk5 and these phosphoepitopes colocalize in neurofibrillary tangles. Adjacent temporal lobe sections were double immunostained with a polyclonal anti-cdk5 and monoclonal AT8 (which recognizes phosphorylated serine 199, serine 202, and threonine 205 in tau) antibodies. A subset of AT8 phosphotau-positive neurons was immunoreactive for cdk5 in entorhinal (area 28) and perirhinal (area 35) cortices and CA1 of the hippocampus. We assessed the ratio of cdk5-positive cells to AT8-positive cells and found that there is a higher degree of colocalization in pre-neurofibrillary tangles as opposed to intraneuronal and extraneuronal neurofibrillary tangles. We further examined colocalization using fluorescence resonance energy transfer. This suggests a close, stable intermolecular association between cdk5 and phosphorylated tau, consistent with phosphorylation of tau by cdk5 in AD brain.

    Funded by: NIA NIH HHS: AG08487; NINDS NIH HHS: NS 07484-01

    Journal of neuropathology and experimental neurology 2002;61;6;557-64

  • Identification of a neuronal Cdk5 activator-binding protein as Cdk5 inhibitor.

    Ching YP, Pang AS, Lam WH, Qi RZ and Wang JH

    Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.

    Neuronal Cdc2-like kinase (Nclk) plays an important role in a variety of cellular processes, including neuronal cell differentiation, apoptosis, neuron migration, and formation of neuromuscular junction. The active kinase consists of a catalytic subunit, Cdk5, and an essential regulatory subunit, neuronal Cdk5 activator (p35(nck5a) or p25(nck5a)), which is expressed primarily in neurons of central nervous tissue. In our previous study using the yeast two-hybrid screening method, three novel p35(nck5a)-associated proteins were isolated. Here we show that one of these proteins, called C42, specifically inhibits the activation of Cdk5 by Nck5a. Co-immunoprecipitation data suggested that C42 and p35(nck5a) could form a complex within cultured mammalian cells. Deletion analysis has mapped the inhibitory domain of C42 to a region of 135 amino acids, which is conserved in Pho81, a yeast protein that inhibits the yeast cyclin-dependent protein kinase Pho85. The Pho85.Pho80 kinase complex has been shown to be the yeast functional homologue of the mammalian Cdk5/p35(nck5a) kinase.

    The Journal of biological chemistry 2002;277;18;15237-40

  • Cyclin-dependent kinase-5/p35 phosphorylates Presenilin 1 to regulate carboxy-terminal fragment stability.

    Lau KF, Howlett DR, Kesavapany S, Standen CL, Dingwall C, McLoughlin DM and Miller CC

    Department of Neuroscience, The Institute of Psychiatry, King's College London, De Crespigny Park, Denmark Hill, United Kingdom.

    Mutations in the Presenilin 1 gene are the cause of the majority of autosomal dominant familial forms of Alzheimer's disease. Presenilin 1 (PS1) is produced as a holoprotein but is then rapidly processed to amino- (N-PS1) and carboxy-terminal (C-PS1) fragments that are incorporated into stable high molecular mass complexes. The mechanisms that control PS1 cleavage and stability are not properly understood but sequences within C-PS1 have been shown to regulate both of these properties. Here we demonstrate that cyclin dependent kinase-5/p35 (cdk5/p35) phosphorylates PS1 on threonine(354) within C-PS1 both in vitro and in vivo. Threonine(354) phosphorylation functions to selectively stabilize C-PS1. Our results demonstrate that cdk5/p35 is a regulator of PS1 metabolism.

    Molecular and cellular neurosciences 2002;20;1;13-20

  • Cdk5 phosphorylates p53 and regulates its activity.

    Zhang J, Krishnamurthy PK and Johnson GV

    Department of Psychiatry and Neurobiology, University of Alabama at Birmingham, 35294-0017, USA.

    Cyclin dependent kinase 5 (Cdk5) is a proline-direct protein kinase that is most active in the CNS, and has been implicated as a contributing factor in certain neurodegenerative diseases. Further, there is evidence to suggest that Cdk5 may facilitate the progression of apoptosis. However, the mechanisms involved have not been elucidated. The tumor suppressor protein p53, a transcription factor that is regulated by phosphorylation, increases the expression of genes that control growth arrest or cell death. To understand how Cdk5 could facilitate apoptosis, the effects of Cdk5 on p53 activity were examined. In the present study it is shown that in apoptotic PC12 cells the levels of p53 and Cdk5 increase concomitantly. Further, Cdk5/p25 effectively phosphorylates recombinant p53 in vitro. Transient transfection of Cdk5/p25 into cells results in an increase in p53 levels, as well as the expression of the p53-responsive genes p21 and Bax. Furthermore, evidence is provided that increased Cdk5 activity increases p53 transcriptional activity significantly, suggesting that p53 is modulated in situ by Cdk5. This is the first demonstration that p53 is a substrate of Cdk5, and that Cdk5 can modulate p53 levels and activity.

    Funded by: NINDS NIH HHS: NS35060

    Journal of neurochemistry 2002;81;2;307-13

  • ik3-2, a relative to ik3-1/cables, is associated with cdk3, cdk5, and c-abl.

    Sato H, Nishimoto I and Matsuoka M

    Department of Pharmacology, Keio University School of Medicine, Tokyo, Japan.

    A cDNA coding for ik3-2 (designated as ik3-2 from an interactor-2 with cdk3) was cloned by cross-hybridization with ik3-1 and RT-PCR. Analysis of amino acid sequence indicated that ik3-2 has the C-terminal cyclin-box-like region highly homologous to that of ik3-1 (identity in amino acids: 78%). On the other hand, the remainder of ik3-2 gene is not so similar to that of ik3-1. There are several regions other than the C-terminal cyclin-box-like region that are conserved between ik3-1 and ik3-2. In vivo binding assay indicated that like ik3-1, ik3-2 binds to cdk3, cdk5, and c-abl, although ik3-2 binds to cdk3 weakly as compared with ik3-1. The C-terminal cyclin-box-like region of ik3-2 (123 amino acids) is able to be associated with cdk5. Accordingly, ik3-2 is very similar to ik3-1 concerning its molecular interaction with other molecules, suggesting that ik3-2 function in the same biological field as ik3-1. Northern blot analysis indicated that ik3-2 is expressed ubiquitously all over tissues.

    Biochimica et biophysica acta 2002;1574;2;157-63

  • Calpain-mediated cleavage of the cyclin-dependent kinase-5 activator p39 to p29.

    Patzke H and Tsai LH

    Department of Pathology, Harvard Medical School and the Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA.

    The activity of cyclin-dependent kinase-5 (Cdk5) is tightly regulated by binding of its neuronal activators p35 and p39. Upon neurotoxic insults, p35 is cleaved to p25 by the Ca(2+)-dependent protease calpain. p25 is accumulated in ischemic brains and in brains of patients with Alzheimer's disease. p25 deregulates Cdk5 activity by causing prolonged activation and mislocalization of Cdk5. It is unknown whether p39, which is expressed throughout the adult rat brain, is cleaved by calpain, and whether this contributes to deregulation of Cdk5. Here, we show that calpain cleaved p39 in vitro, resulting in generation of a C-terminal p29 fragment. In vivo, p29 was generated in ischemic brain concomitant with increased calpain activity. In fresh brain lysates, generation of p29 was Ca(2+)-dependent, and calpain inhibitors abolished p29 production. The Ca(2+) ionophore ionomycin and the excitotoxin glutamate induced production of p29 in cultures of cortical neurons in a calpain-dependent manner. Like p25, p29 was more stable than p39 and caused redistribution of Cdk5 in cortical neurons. Our data suggest that neurotoxic insults lead to calpain-mediated conversion of p39 to p29, which might contribute to deregulation of Cdk5.

    Funded by: NIGMS NIH HHS: GM 53049

    The Journal of biological chemistry 2002;277;10;8054-60

  • The protein SET binds the neuronal Cdk5 activator p35nck5a and modulates Cdk5/p35nck5a activity.

    Qu D, Li Q, Lim HY, Cheung NS, Li R, Wang JH and Qi RZ

    Proteomics Group, Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore 117609.

    The neuronal Cdk5 kinase is composed of the catalytic subunit Cdk5 and the activator protein p35(nck5a) or its isoform, p39(nck5ai). To identify novel p35(nck5a)- and p39(nck5ai)-binding proteins, fragments of p35(nck5a) and p39(nck5ai) were utilized in affinity isolation of binding proteins from rat brain homogenates, and the isolated proteins were identified using mass spectrometry. With this approach, the nuclear protein SET was shown to interact with the N-terminal regions of p35(nck5a) and p39(nck5ai). Our detailed characterization showed that the SET protein formed a complex with Cdk5/p35(nck5a) through its binding to p35(nck5a). The p35(nck5a)-interacting region was mapped to a predicted alpha-helix in SET. When cotransfected into COS-7 cells, SET and p35(nck5a) displayed overlapping intracellular distribution in the nucleus. The nuclear co-localization was corroborated by immunostaining data of endogenous SET and Cdk5/p35(nck5a) from cultured cortical neurons. Finally, we demonstrated that the activity of Cdk5/p35(nck5a), but not that of Cdk5/p25(nck5a), was enhanced upon binding to the SET protein. The tail region of SET, which is rich in acidic residues, is required for the stimulatory effect on Cdk5/p35(nck5a).

    The Journal of biological chemistry 2002;277;9;7324-32

  • Increase of cdk5 is related to neurofibrillary pathology in progressive supranuclear palsy.

    Borghi R, Giliberto L, Assini A, Delacourte A, Perry G, Smith MA, Strocchi P, Zaccheo D and Tabaton M

    Department of Neuroscience, University of Genova, Italy.

    Background: Progressive supranuclear palsy (PSP) is characterized by a pure neurofibrillary tau pathology involving mainly basal ganglia and brainstem nuclei. In addition to a haplotype of the tau gene potentially favoring tau aggregation, lipoperoxidation has been shown to be associated with PSP tau pathology.

    Objective: To analyze cdk5/p35 complex, a kinase that regulates neurite outgrowth, as a potential cellular mechanism underlying tau phosphorylation in brain tissues from PSP and control cases and comparatively in cerebral cortex from subjects with AD.

    Methods: Cdk5/p35 protein levels and distribution were evaluated by immunoblotting and immunocytochemistry in brain regions from seven PSP, six AD, and seven control cases, with similar postmortem intervals.

    Results: Total cdk5 protein levels were significantly increased by more than threefold in PSP tissue and were augmented in PSP neurons, codistributed with tau immunoreactivity. P35, the regulatory subunit of cdk5, was degraded by postmortem proteolysis to the same extent in PSP, AD, and control tissues.

    Conclusions: The proteolysis in vivo of p35, the regulatory subunit of the kinase, is not ascertainable because it is masked by its postmortem degradation. The study, however, indicates that in PSP, the alteration of cdk5 is different from that described in AD and suggests that the absence of amyloid beta protein deposition may account for the different pathways responsible for the same kinase activation.

    Funded by: NINDS NIH HHS: NS38648

    Neurology 2002;58;4;589-92

  • Cyclin-dependent kinase 5 prevents neuronal apoptosis by negative regulation of c-Jun N-terminal kinase 3.

    Li BS, Zhang L, Takahashi S, Ma W, Jaffe H, Kulkarni AB and Pant HC

    Laboratory of Neurochemistry, NINDS, NIH, Bethesda, MD 20892-4130, USA.

    Cyclin-dependent kinase 5 (cdk5) is a serine/threonine kinase activated by associating with its neuron-specific activators p35 and p39. Analysis of cdk5(-/-) and p35(-/-) mice has demonstrated that both cdk5 and p35 are essential for neuronal migration, axon pathfinding and the laminar configuration of the cerebral cortex, suggesting that the cdk5-p35 complex may play a role in neuron survival. However, the targets of cdk5 that regulate neuron survival are unknown. Here, we show that cdk5 directly phosphorylates c-Jun N-terminal kinase 3 (JNK3) on Thr131 and inhibits its kinase activity, leading to reduced c-Jun phosphorylation. Expression of cdk5 and p35 in HEK293T cells inhibits c-Jun phosphorylation induced by UV irradiation. These effects can be restored by expression of a catalytically inactive mutant form of cdk5. Moreover, cdk5-deficient cultured cortical neurons exhibit increased sensitivity to apoptotic stimuli, as well as elevated JNK3 activity and c-Jun phosphorylation. Taken together, these findings show that cdk5 may exert its role as a key element by negatively regulating the c-Jun N-terminal kinase/stress-activated protein kinase signaling pathway during neuronal apoptosis.

    The EMBO journal 2002;21;3;324-33

  • Phosphorylation of MEK1 by cdk5/p35 down-regulates the mitogen-activated protein kinase pathway.

    Sharma P, Veeranna, Sharma M, Amin ND, Sihag RK, Grant P, Ahn N, Kulkarni AB and Pant HC

    Laboratory of Neurochemistry, NINDS, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Cyclin-dependent protein kinase 5 (cdk5), a member of the cdk family, is active mainly in postmitotic cells and plays important roles in neuronal development and migration, neurite outgrowth, and synaptic transmission. In this study we investigated the relationship between cdk5 activity and regulation of the mitogen-activated protein (MAP) kinase pathway. We report that cdk5 phosphorylates the MAP kinase kinase-1 (MEK1) in vivo as well as the Ras-activated MEK1 in vitro. The phosphorylation of MEK1 by cdk5 resulted in inhibition of MEK1 catalytic activity and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. In p35 (cdk5 activator) -/- mice, which lack appreciable cdk5 activity, we observed an increase in the phosphorylation of NF-M subunit of neurofilament proteins that correlated with an up-regulation of MEK1 and ERK1/2 activity. The activity of a constitutively active MEK1 with threonine 286 mutated to alanine (within a TPXK cdk5 phosphorylation motif in the proline-rich domain) was not affected by cdk5 phosphorylation, suggesting that Thr286 might be the cdk5/p35 phosphorylation-dependent regulatory site. These findings support the hypothesis that cdk5 and the MAP kinase pathway cross-talk in the regulation of neuronal functions. Moreover, these data and the recent studies of Harada et al. (Harada, T., Morooka, T., Ogawa, S., and Nishida, E. (2001) Nat. Cell Biol. 3, 453-459) have prompted us to propose a model for feedback down-regulation of the MAP kinase signal cascade by cdk5 inactivation of MEK1.

    The Journal of biological chemistry 2002;277;1;528-34

  • Aberrant glycosylation modulates phosphorylation of tau by protein kinase A and dephosphorylation of tau by protein phosphatase 2A and 5.

    Liu F, Zaidi T, Iqbal K, Grundke-Iqbal I and Gong CX

    Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, , Staten Island, NY 10314, USA.

    Microtubule-associated protein tau is abnormally hyperphosphorylated, glycosylated, and aggregated in affected neurons in Alzheimer's disease (AD). We recently found that the aberrant tau glycosylation precedes tau hyperphosphorylation in AD brain. In the present study, we developed assays to determine phosphorylation and dephosphorylation of tau at specific phosphorylation sites by using glycosylated tau purified from AD brain as a substrate. We then studied the effects of the aberrant glycosylation on phosphorylation and dephosphorylation of tau at each specific phosphorylation site. We found that deglycosylation of the aberrantly glycosylated tau decreased the subsequent phosphorylation of tau at Ser214, Ser262, and Ser356 in vitro by protein kinase A. On the other hand, deglycosylation of tau positively modulated the subsequent dephosphorylation by protein phosphatase 2A and protein phosphatase 5 in vitro at the phosphorylation sites Ser198, Ser199, and Ser202. Our results suggest that the aberrant glycosylation may modulate tau protein at a substrate level so that it is easier to be phosphorylated and more difficult to be dephosphorylated at some phosphorylation sites in AD brain. The combined impact of this modulation may be to make tau more susceptible to becoming abnormally hyperphosphorylated.

    Funded by: NIA NIH HHS: AG05892, AG08076, AG16760; NINDS NIH HHS: MN/NS31862, NS18105

    Neuroscience 2002;115;3;829-37

  • Phosphorylation of Pak1 by the p35/Cdk5 kinase affects neuronal morphology.

    Rashid T, Banerjee M and Nikolic M

    Molecular and Developmental Neurobiology Medical Research Council Centre, New Hunt's House, King's College London, London, SE1 1UL, United Kingdom.

    The small GTPase Rac and its effectors, the Pak1 and p35/Cdk5 kinases, have been assigned important roles in regulating cytoskeletal dynamics in neurons. Our previous work revealed that the neuronal p35/Cdk5 kinase associates with Pak1 in a RacGTP-dependent manner, causing hyperphosphorylation and down-regulation of Pak1 kinase activity. We have now demonstrated direct phosphorylation of Pak1 on threonine 212 by the p35/Cdk5 kinase. In neuronal growth cones, Pak1 phosphorylated on Thr-212 localized to actin and tubulin-rich areas, suggesting a role in regulating growth cone dynamics. The expression of a non-phosphorylatable Pak1 mutant (Pak1A212) induced dramatic neurite disorganization. We also observed a strong association between p35/Cdk5 and the Pak1 C-terminal kinase domain. Overall, our data show that in neurons, membrane-associated, active Pak1 is regulated by the p35/Cdk5 kinase both by association and phosphorylation, which is essential for the proper regulation of the cytoskeleton during neurite outgrowth and remodeling.

    The Journal of biological chemistry 2001;276;52;49043-52

  • The Cdk5-p35 kinase associates with the Golgi apparatus and regulates membrane traffic.

    Paglini G, Peris L, Diez-Guerra J, Quiroga S and Cáceres A

    INIMEC-CONICET, Avenue Friuli 2434, 5016 Córdoba, Argentina.

    We show here that an active Cdk5-p35 kinase is present in Golgi membranes, where it associates with a detergent-insoluble fraction containing actin. In addition, Cdk5-p35-dependent phosphorylation of alpha-PAK immunoreactive protein species was detected in Golgi membranes, as well as an interaction with the small GTPase, Cdc42. Moreover, antisense oligonucleotide suppression of Cdk5 or p35 in young cultured neurons, as well as inhibition of Cdk5 activity with olomoucine, blocks the formation of membrane vesicles from the Golgi apparatus. Taken together, these results show a novel subcellular localization of this kinase and suggest a role for Cdk5-p35 in membrane traffic during neuronal process outgrowth.

    EMBO reports 2001;2;12;1139-44

  • ik3-1/Cables is a substrate for cyclin-dependent kinase 3 (cdk 3).

    Yamochi T, Semba K, Tsuji K, Mizumoto K, Sato H, Matsuura Y, Nishimoto I and Matsuoka M

    Department of Pharmacology, KEIO University School of Medicine, Tokyo, Japan.

    p70ik3-1 (a 70-kDa protein) contains a cyclin box, and binds to p35cdk3 in vivo and in vitro [Matsuoka, M., Matsuura, Y., Semba, K. & Nishimoto, I. (2000) Biochem. Biophys. Res. Commun. 273, 442-447]. In spite of its structural similarity to cyclins, p70ik3-1 does not activate cyclin-dependent kinase 3 (cdk3)-mediated phosphorylation of pRb, histone H1, or the C-terminal domain of RNA polymerase II. Here, we report that Ser274 of p70ik3-1 is phosphorylated by cdk2 or cdk3 bound to cyclin A and to cyclin E in vitro. We also found that in COS7 cells in which cyclin E and cdk3 were ectopically overexpressed, the phosphorylation level of Ser274 in coexpressed p70ik3-1 is upregulated. We therefore conclude that p70ik3-1 is a substrate for cdk3-mediated phosphorylation.

    European journal of biochemistry 2001;268;23;6076-82

  • Regulation of NMDA receptors by cyclin-dependent kinase-5.

    Li BS, Sun MK, Zhang L, Takahashi S, Ma W, Vinade L, Kulkarni AB, Brady RO and Pant HC

    Laboratory of Neurochemistry, Laboratory of Adaptive Systems, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.

    Members of the N-methyl-d-aspartate (NMDA) class of glutamate receptors (NMDARs) are critical for development, synaptic transmission, learning and memory; they are targets of pathological disorders in the central nervous system. NMDARs are phosphorylated by both serine/threonine and tyrosine kinases. Here, we demonstrate that cyclin dependent kinase-5 (Cdk5) associates with and phosphorylates NR2A subunits at Ser-1232 in vitro and in intact cells. Moreover, we show that roscovitine, a selective Cdk5 inhibitor, blocks both long-term potentiation induction and NMDA-evoked currents in rat CA1 hippocampal neurons. These results suggest that Cdk5 plays a key role in synaptic transmission and plasticity through its up-regulation of NMDARs.

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;22;12742-7

  • Structure and regulation of the CDK5-p25(nck5a) complex.

    Tarricone C, Dhavan R, Peng J, Areces LB, Tsai LH and Musacchio A

    Structural Biology Unit, Department of Experimental Oncology, European Institute of Oncology, Via Ripamonti 435, I-20141 Milan, Italy.

    CDK5 plays an indispensable role in the central nervous system, and its deregulation is involved in neurodegeneration. We report the crystal structure of a complex between CDK5 and p25, a fragment of the p35 activator. Despite its partial structural similarity with the cyclins, p25 displays an unprecedented mechanism for the regulation of a cyclin-dependent kinase. p25 tethers the unphosphorylated T loop of CDK5 in the active conformation. Residue Ser159, equivalent to Thr160 on CDK2, contributes to the specificity of the CDK5-p35 interaction. Its substitution with threonine prevents p35 binding, while the presence of alanine affects neither binding nor kinase activity. Finally, we provide evidence that the CDK5-p25 complex employs a distinct mechanism from the phospho-CDK2-cyclin A complex to establish substrate specificity.

    Molecular cell 2001;8;3;657-69

  • Neuronal Cdc2-like protein kinase (Cdk5/p25) is associated with protein phosphatase 1 and phosphorylates inhibitor-2.

    Agarwal-Mawal A and Paudel HK

    Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Quebec, H3T 1E2, Canada.

    Protein phosphatase 1 (PP1) is complexed with inhibitor 2 (I-2) in the cytosol. In rabbit muscle extract PP1.I-2 is activated upon preincubation with ATP/Mg. This activation is caused by phosphorylation of I-2 on Thr(72) by glycogen synthase kinase 3 (GSK3). We have found that PP1.I-2 in bovine brain extract is also activated upon preincubation with ATP/Mg. However, blocking GSK3 action by LiCl inhibited only approximately 29% of PP1 activity and indicated that GSK3 is not the sole PP1.I-2 activator in the brain. When bovine brain extract was analyzed by gel filtration PP1.I-2 and neuronal Cdc2-like protein kinase (NCLK), a heterodimer of Cdk5 and the regulatory p25 subunit, co-eluted as a approximately 450-kDa size species. The NCLK from the eluted column fractions bound to PP1-specific microcystin-Sepharose and glutathione S-transferase (GST)-I-2-coated glutathione-agarose beads. Similarly, PP1 from the eluted column fractions was pulled down with GST-Cdk5-coated glutathione-agarose beads. In vitro, NCLK phosphorylated I-2 on Thr(72) and activated PP1.I-2 in an ATP/Mg-dependent manner. NCLK bound to PP1 through its Cdk5 subunit and the PP1 binding region was localized to Cdk5 residues 28-41. Our data demonstrate that in brain extract PP1.I-2 and NCLK are associated within a complex of approximately 450 kDa and suggest that NCLK is one of the PP1.I-2-activating kinases in the mammalian brain.

    The Journal of biological chemistry 2001;276;26;23712-8

  • Expression of the neuronal cyclin-dependent kinase 5 activator p35Nck5a in human monocytic cells is associated with differentiation.

    Chen F and Studzinski GP

    Department of Pathology and Laboratory Medicine, UMDNJ-New Jersey Medical School, 185 S Orange Ave., Newark, NJ 07103, USA.

    Although cyclin-dependent kinase 5 (Cdk5) is widely expressed in human tissues, its activator p35Nck5a is generally considered to be neuron specific. In addition to neuronal cells, active Cdk5 complexes have been reported in developing tissues, such as the embryonic muscle and ocular lens, and in human leukemia HL60 cells induced to differentiate by an exposure to 1,25-dihydroxyvitamin D(3); however, its activator in these cells has not been demonstrated. The results of this study indicate that p35Nck5a is associated with Cdk5 in monocytic differentiation of hematopoietic cells. Specifically, p35Nck5a is expressed in normal human monocytes and in leukemic cells induced to differentiate toward the monocytic lineage, but not in lymphocytes or cells induced to granulocytic differentiation by retinoic acid. It is present in a complex with Cdk5 that has protein kinase activity, and when ectopically expressed together with Cdk5 in undifferentiated HL60 cells, it induces the expression of CD14 and "nonspecific" esterase, markers of monocytic phenotype. These observations not only indicate a functional relationship between Cdk5 and p35Nck5a, but also support a role for this complex in monocytic differentiation. (Blood. 2001;97:3763-3767)

    Funded by: NCI NIH HHS: 2R01-CA44722

    Blood 2001;97;12;3763-7

  • Mitotic reorganization of the intermediate filament protein nestin involves phosphorylation by cdc2 kinase.

    Sahlgren CM, Mikhailov A, Hellman J, Chou YH, Lendahl U, Goldman RD and Eriksson JE

    Turku Centre for Biotechnology, University of Turku and Abo Akademi University, FIN-20521 Turku, Finland.

    The intermediate filament protein nestin is expressed during early stages of development in the central nervous system and in muscle tissues. Nestin expression is associated with morphologically dynamic cells, such as dividing and migrating cells. However, little is known about regulation of nestin during these cellular processes. We have characterized the phosphorylation-based regulation of nestin during different stages of the cell cycle in a neuronal progenitor cell line, ST15A. Confocal microscopy of nestin organization and (32)P in vivo labeling studies show that the mitotic reorganization of nestin is accompanied by elevated phosphorylation of nestin. The phosphorylation-induced alterations in nestin organization during mitosis in ST15A cells are associated with partial disassembly of nestin filaments. Comparative in vitro and in vivo phosphorylation studies identified cdc2 as the primary mitotic kinase and Thr(316) as a cdc2-specific phosphorylation site on nestin. We generated a phosphospecific nestin antibody recognizing the phosphorylated form of this site. By using this antibody we observed that nestin shows constitutive phosphorylation at Thr(316), which is increased during mitosis. This study shows that nestin is reorganized during mitosis and that cdc2-mediated phosphorylation is an important regulator of nestin organization and dynamics during mitosis.

    The Journal of biological chemistry 2001;276;19;16456-63

  • The kinase DYRK phosphorylates protein-synthesis initiation factor eIF2Bepsilon at Ser539 and the microtubule-associated protein tau at Thr212: potential role for DYRK as a glycogen synthase kinase 3-priming kinase.

    Woods YL, Cohen P, Becker W, Jakes R, Goedert M, Wang X and Proud CG

    MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, Dow Street, University of Dundee, Dundee DD1 5EH, Scotland, UK. y.l.woods@dundee.ac.uk

    The substrate specificity of glycogen synthase kinase 3 (GSK3) is unusual in that efficient phosphorylation only occurs if another phosphoserine or phosphothreonine residue is already present four residues C-terminal to the site of GSK3 phosphorylation. One such substrate is the epsilon-subunit of rat eukaryotic protein-synthesis initiation factor 2B (eIF2Bepsilon), which is inhibited by the GSK3-catalysed phosphorylation of Ser(535). There is evidence that GSK3 is only able to phosphorylate eIF2Bepsilon at Ser(535) if Ser(539) is already phosphorylated by another protein kinase. However, no protein kinases capable of phosphorylating Ser(539) have so far been identified. Here we show that Ser(539) of eIF2Bepsilon, which is followed by proline, is phosphorylated specifically by two isoforms of dual-specificity tyrosine phosphorylated and regulated kinase (DYRK2 and DYRK1A), but only weakly or not at all by other 'proline-directed' protein kinases tested. We also establish that phosphorylation of Ser(539) permits GSK3 to phosphorylate Ser(535) in vitro and that eIF2Bepsilon is highly phosphorylated at Ser(539) in vivo. The DYRK isoforms also phosphorylate human microtubule-associated protein tau at Thr(212) in vitro, a residue that is phosphorylated in foetal tau and hyperphosphorylated in filamentous tau from Alzheimer's-disease brain. Phosphorylation of Thr(212) primes tau for phosphorylation by GSK3 at Ser(208) in vitro, suggesting a more general role for DYRK isoforms in priming phosphorylation of GSK3 substrates.

    The Biochemical journal 2001;355;Pt 3;609-15

  • Reactivity of anti-proliferating cell nuclear antigen (PCNA) murine monoclonal antibodies and human autoantibodies to the PCNA multiprotein complexes involved in cell proliferation.

    Takasaki Y, Kogure T, Takeuchi K, Kaneda K, Yano T, Hirokawa K, Hirose S, Shirai T and Hashimoto H

    Department of Internal Medicine and Rheumatology, Juntendo University School of Medicine, Tokyo, Japan. tyoshi@med.juntendo.ac.jp

    Proliferating cell nuclear Ag (PCNA) occurs as a component of multiprotein complexes during cell proliferation. We found the complexes to react with murine anti-PCNA mAbs, but not with anti-PCNA Abs in lupus sera. The complexes were purified from rabbit thymus extract by affinity chromatography using anti-PCNA mAbs (TOB7, TO17, and TO30) and analyzed by ELISA, immunoprecipitation, immunoblotting, and HPLC gel filtration. That PCNA was complexed with other proteins was demonstrated by its copurification with a group of proteins excluded by an HPLC G3000 SW column. Although immunoblot analysis showed the mAbs to react exclusively with the 34-kDa PCNA polypeptide, they nonetheless immunoprecipitated the same group of proteins, confirming the interaction of the isolated PCNA with other proteins. Anti-PCNA sera, including AK, which reacts with biologically functional sites on PCNA, did not react with complexed PCNA, but did react with it once it was dissociated from the complexes. PCNA complexes in turn reacted with murine anti-DNA mAbs, as well as with Abs against p21, replication protein A, DNA helicase II, cyclin-dependent kinases 4 and 5, and topoisomerase I. These findings suggest that the PCNA complexes purified using anti-PCNA mAbs comprise the "protein machinery" for DNA replication and cell cycle regulation. They also suggest that anti-PCNA mAbs are useful tools with which to characterize the protein-protein interactions within PCNA complexes, as well as the autoimmune responses to proteins interacting with PCNA, which may shed light on the mechanisms of autoantibody production in lupus patients.

    Journal of immunology (Baltimore, Md. : 1950) 2001;166;7;4780-7

  • Phosphorylation of tau is regulated by PKN.

    Taniguchi T, Kawamata T, Mukai H, Hasegawa H, Isagawa T, Yasuda M, Hashimoto T, Terashima A, Nakai M, Mori H, Ono Y and Tanaka C

    Hyogo Institute for Aging Brain and Cognitive Disorders, Himeji 670-0981, Japan. tanigu@hiabcd.go.jp

    For the phosphorylation state of microtubule-associated protein, tau plays a pivotal role in regulating microtubule networks in neurons. Tau promotes the assembly and stabilization of microtubules. The potential for tau to bind to microtubules is down-regulated after local phosphorylation. When we investigated the effects of PKN activation on tau phosphorylation, we found that PKN triggers disruption of the microtubule array both in vitro and in vivo and predominantly phosphorylates tau in microtubule binding domains (MBDs). PKN has a catalytic domain highly homologous to protein kinase C (PKC), a kinase that phosphorylates Ser-313 (= Ser-324, the number used in this study) in MBDs. Thus, we identified the phosphorylation sites of PKN and PKC subtypes (PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, -zeta, and -lambda) in MBDs. PKN phosphorylates Ser-258, Ser-320, and Ser-352, although all PKC subtypes phosphorylate Ser-258, Ser-293, Ser-324, and Ser-352. There is a PKN-specific phosphorylation site, Ser-320, in MBDs. HIA3, a novel phosphorylation-dependent antibody recognizing phosphorylated tau at Ser-320, showed immunoreactivity in Chinese hamster ovary cells expressing tau and the active form of PKN, but not in Chinese hamster ovary cells expressing tau and the inactive form of PKN. The immunoreactivity for phosphorylated tau at Ser-320 increased in the presence of a phosphatase inhibitor, FK506 treatment, which means that calcineurin (protein phosphatase 2B) may be involved in dephosphorylating tau at Ser-320 site. We also noted that PKN reduces the phosphorylation recognized by the phosphorylation-dependent antibodies AT8, AT180, and AT270 in vivo. Thus PKN serves as a regulator of microtubules by specific phosphorylation of tau, which leads to disruption of tubulin assembly.

    The Journal of biological chemistry 2001;276;13;10025-31

  • Amphiphysin 1 binds the cyclin-dependent kinase (cdk) 5 regulatory subunit p35 and is phosphorylated by cdk5 and cdc2.

    Floyd SR, Porro EB, Slepnev VI, Ochoa GC, Tsai LH and De Camilli P

    Howard Hughes Medical Institute and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

    Amphiphysin 1 is a phosphoprotein expressed at high levels in neurons, where it participates in synaptic vesicle endocytosis and neurite outgrowth. It is a substrate for cyclin-dependent kinase (cdk) 5, a member of the cyclin-dependent protein kinase family, which has been functionally linked to neuronal migration and neurite outgrowth via its action on the actin cytoskeleton. The yeast homologue of amphiphysin, Rvs167, functions in endocytosis and actin dynamics, is phosphorylated by the cdk5 homologue Pho85, and binds the Pho85 regulatory subunit Pcl2. We show here that amphiphysin 1 interacts with the cdk5-activating subunit p35 and that this interaction is mediated by the conserved NH2-terminal region of amphiphysin. Amphiphysin 1 colocalizes with p35 in the growth cones of neurons and at actin-rich peripheral lamellipodia in transfected fibroblasts. Amphiphysin is phosphorylated by cdk5 in a region including serines 272, 276, and 285. Amphiphysin 1 is also phosphorylated by the cdc2/cyclin B kinase complex in the same region and undergoes mitotic phosphorylation in dividing cells. These data indicate that phosphorylation by members of the cyclin-dependent kinase family is a conserved property of amphiphysin and suggest that this phosphorylation may play an important physiological role both in mitosis and in differentiated cells.

    Funded by: NCI NIH HHS: CA46128; NINDS NIH HHS: NS36251, NS37007

    The Journal of biological chemistry 2001;276;11;8104-10

  • Characterization of the in vitro phosphorylation of human tau by tau protein kinase II (cdk5/p20) using mass spectrometry.

    Lund ET, McKenna R, Evans DB, Sharma SK and Mathews WR

    Structural, Analytical and Medicinal Chemistry, Pharmacia Corporation, Kalamazoo, USA.

    Hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies, and tau protein kinase II is reported to play a role in the pathogenesis of Alzheimer's disease. Recently, we reported that tau protein kinase II (cdk5/p20)-phosphorylated human tau inhibits microtubule assembly, and tau protein kinase II (cdk5/p20) phosphorylation of microtubule-associated tau results in dissociation of phosphorylated tau from the microtubules and tubulin depolymerization. In the studies reported here, a combination of mass spectrometric techniques was used to study the phosphorylation of human recombinant tau by recombinant tau protein kinase II (cdk5/p20) in vitro. The extent of phosphorylation was determined by measuring the molecular mass of phosphorylated tau using mass spectrometry. Reaction of human recombinant tau with tau protein kinase II (cdk5/p20) resulted in the formation of two major species containing either five or six phosphate groups. The specific amino acid residues phosphorylated were determined by analyzing tryptic peptides by tandem mass spectrometry via either MALDI/TOF post-source decay or by electrospray tandem mass spectrometry. Based on these experiments, we conclude that tau protein kinase II (cdk5/p20) can phosphorylate human tau at Thr(181), Thr(205), Thr(212), Thr(217), Ser(396) and Ser(404).

    Journal of neurochemistry 2001;76;4;1221-32

  • p35/cdk5 binds and phosphorylates beta-catenin and regulates beta-catenin/presenilin-1 interaction.

    Kesavapany S, Lau KF, McLoughlin DM, Brownlees J, Ackerley S, Leigh PN, Shaw CE and Miller CC

    Department of Neuroscience, The Institute of Psychiatry, Denmark Hill, London SE5 8AF, UK.

    The neuronal cyclin-dependent kinase p35/cdk5 comprises a catalytic subunit (cdk5) and an activator subunit (p35). To identify novel p35/cdk5 substrates, we utilized the yeast two-hybrid system to screen for human p35 binding partners. From one such screen, we identified beta-catenin as an interacting protein. Confirmation that p35 binds to beta-catenin was obtained by using glutathione S-transferase (GST)-beta-catenin fusion proteins that interacted with both endogenous and transfected p35, and by showing that beta-catenin was present in p35 immunoprecipitates. p35 and beta-catenin also displayed overlapping subcellular distribution patterns in cells including neurons. Finally, we demonstrated that p35/cdk5 phosphorylates beta-catenin. beta-catenin also binds to presenilin-1 and altered beta-catenin/presenilin-1 interactions may be mechanistic in Alzheimer's disease (AD). Abnormal p35/cdk5 activity has also been suggested to contribute to AD. We therefore investigated how modulation of p35/cdk5 activity influenced beta-catenin/presenilin-1 interactions. Inhibition of p35/cdk5 with roscovitine did not alter the steady state levels of either beta-catenin or presenilin-1 but reduced the amount of presenilin-1 bound to beta-catenin. Thus, p35/cdk5 binds and phosphorylates beta-catenin and regulates its binding to presenilin-1. The findings reported here therefore provide a novel molecular framework to connect p35/cdk5 with beta-catenin and presenilin-1 in AD.

    The European journal of neuroscience 2001;13;2;241-7

  • Glycogen synthase kinase3 beta phosphorylates serine 33 of p53 and activates p53's transcriptional activity.

    Turenne GA and Price BD

    Department of Radiation Oncology, D810A, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney St, Boston, MA 02115, USA. aturenne@kendallstrategies.com

    Background: The p53 protein is activated by genotoxic stress, oncogene expression and during senescence, p53 transcriptionally activates genes involved in growth arrest and apoptosis. p53 activation is regulated by post-translational modification, including phosphorylation of the N-terminal transactivation domain. Here, we have examined how Glycogen Synthase Kinase (GSK3), a protein kinase involved in tumorigenesis, differentiation and apoptosis, phosphorylates and regulates p53.

    Results: The 2 isoforms of GSK3, GSK3alpha and GSK3beta, phosphorylate the sequence Ser-X-X-X-Ser(P) when the C-terminal serine residue is already phosphorylated. Several p53 kinases were examined for their ability to create GSK3 phosphorylation sites on the p53 protein. Our results demonstrate that phosphorylation of serine 37 of p53 by DNA-PK creates a site for GSK3beta phosphorylation at serine 33 in vitro. GSK3alpha did not phosphorylate p53 under any condition. GSK3beta increased the transcriptional activity of the p53 protein in vivo. Mutation of either serine 33 or serine 37 of p53 to alanine blocked the ability of GSK3beta to regulate p53 transcriptional activity. GSK3beta is therefore able to regulate p53 function in vivo. p53's transcriptional activity is commonly increased by DNA damage. However, GSK3beta kinase activity was inhibited in response to DNA damage, suggesting that GSK3beta regulation of p53 is not involved in the p53-DNA damage response.

    Conclusions: GSK3beta can regulate p53's transcriptional activity by phosphorylating serine 33. However, GSK3beta does not appear to be part of the p53-DNA damage response pathway. Instead, GSK3beta may provide the link between p53 and non-DNA damage mechanisms for p53 activation.

    Funded by: NCI NIH HHS: CA64585, R01 CA064585

    BMC cell biology 2001;2;12

  • NUDEL is a novel Cdk5 substrate that associates with LIS1 and cytoplasmic dynein.

    Niethammer M, Smith DS, Ayala R, Peng J, Ko J, Lee MS, Morabito M and Tsai LH

    Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachussetts 02115, USA.

    Disruption of one allele of the LIS1 gene causes a severe developmental brain abnormality, type I lissencephaly. In Aspergillus nidulans, the LIS1 homolog, NUDF, and cytoplasmic dynein are genetically linked and regulate nuclear movements during hyphal growth. Recently, we demonstrated that mammalian LIS1 regulates dynein functions. Here we characterize NUDEL, a novel LIS1-interacting protein with sequence homology to gene products also implicated in nuclear distribution in fungi. Like LIS1, NUDEL is robustly expressed in brain, enriched at centrosomes and neuronal growth cones, and interacts with cytoplasmic dynein. Furthermore, NUDEL is a substrate of Cdk5, a kinase known to be critical during neuronal migration. Inhibition of Cdk5 modifies NUDEL distribution in neurons and affects neuritic morphology. Our findings point to cross-talk between two prominent pathways that regulate neuronal migration.

    Neuron 2000;28;3;697-711

  • Identification of a common protein association region in the neuronal Cdk5 activator.

    Wang X, Ching YP, Lam WH, Qi Z, Zhang M and Wang JH

    Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, Peoples Republic of China.

    Cyclin-dependent protein kinase 5 (Cdk5) depends on the association with neuronal Cdk5 activator (Nck5a) for kinase activity. A variety of cellular proteins have been shown to undergo high affinity association with Nck5a, including three novel proteins, C42, C48, and C53 found by a yeast two-hybrid screen (Ching, Y. P., Qi, Z., and Wang, J. H. (2000) Gene 242, 285-294). The three proteins show competitive binding to Nck5a suggesting that they bind at a common site. The binding site has been mapped to a region of 26 amino acid residues (residues 145 to 170) at the N-terminal boundary of the kinase activation domain of Nck5a. This region of Nck5a contains an amphipathic alpha-helix whose hydrophobic face is involved in Cdk5 activation (Chin, K. T., Ohki, S, Tang, D., Cheng, H. C., Wang, J. H. , and Zhang, M. (1999) J. Biol. Chem. 274, 7120-7127). Several lines of evidence suggest that Nck5a interacts with the binding proteins at the hydrophilic face of the amphipathic alpha-helix. First, the Nck5a-(145-170) peptide can bind Cdk5 and Nck5a-binding proteins simultaneously. Second, the association of Nck5a-(145-170) to C48 can be markedly reduced by high ionic strength whereas the interaction between Nck5a and Cdk5 is not affected. Third, substitution of Glu(157) by glutamine in Nck5a-(145-170) abolishes the peptide's ability to bind to the three Nck5a-binding proteins without diminishing its Cdk5 binding activity.

    The Journal of biological chemistry 2000;275;41;31763-9

  • CROC-4: a novel brain specific transcriptional activator of c-fos expressed from proliferation through to maturation of multiple neuronal cell types.

    Jeffrey PL, Capes-Davis A, Dunn JM, Tolhurst O, Seeto G, Hannan AJ and Lin SL

    Developmental Neurobiology Unit, Children's Medical Research Institute, Westmead, NSW, 2145, Australia. pjeffrey@cmri.usyd.edu.au

    A novel, brain-specific cDNA, denoted CROC-4, was cloned from human brain by a contingent replication of cDNA procedure capable of detecting transcriptional activators of the human c-fos proto-oncogene promoter. CROC-4 encoded an 18-kDa serine/threonine-rich polypeptide containing a P-loop motif and an SH3-binding region with phosphorylation sites for a variety of protein kinases (cdc2, CDK2, MAPK, CDK5, protein kinase C, Ca(2+)/calmodulin protein kinase 2, casein kinase 2) involved in cell proliferation and differentiation. Immunohistochemistry revealed that during early development, expression was associated with proliferating and migrating cells throughout the rodent brain, initially appearing in the proliferative ventricular zones. During late development and in adult human brain, CROC-4 was expressed in diverse brain regions including the thalamus, subthalamic nucleus, corpus callosum, substantia nigra, caudate nucleus, amygdala, and hippocampus. The association of CROC-4 expression with proliferating regions of developing brain and retention in regions of the adult brain, as well as the punctate nuclear location, suggest that CROC-4 participates in brain-specific c-fos signaling pathways involved in cellular remodeling of brain architecture.

    Molecular and cellular neurosciences 2000;16;3;185-96

  • Neuron-specific phosphorylation of Alzheimer's beta-amyloid precursor protein by cyclin-dependent kinase 5.

    Iijima K, Ando K, Takeda S, Satoh Y, Seki T, Itohara S, Greengard P, Kirino Y, Nairn AC and Suzuki T

    Laboratory of Neurobiophysics, School of Pharmaceutical Sciences, The University of Tokyo, Japan.

    The mature form of Alzheimer's beta-amyloid precursor protein (APP) is phosphorylated specifically at Thr(668) in neurons. In mature neurons, phosphorylated APP is detected in neurites, with dephosphorylated APP being found mostly in the cell body. In vitro, active cyclin-dependent kinase 5 (Cdk5) phosphorylated the cytoplasmic domain of APP at Thr(668). Treatment of mature neurons with an antisense oligonucleotide to Cdk5 suppressed Cdk5 expression and significantly diminished the level of phosphorylated APP. The expression of APP was unaffected in antisense-treated neurons. These results indicate that in neurons APP is phosphorylated by Cdk5, and that this may play a role in its localization.

    Funded by: NIA NIH HHS: AG09464

    Journal of neurochemistry 2000;75;3;1085-91

  • The C-terminal regulatory domain of p53 contains a functional docking site for cyclin A.

    Luciani MG, Hutchins JR, Zheleva D and Hupp TR

    Department of Molecular and Cellular Pathology, Cancer Research Campaign Laboratories, University of Dundee Medical School, Scotland.

    Radiation injury to cells enhances C-terminal phosphorylation of p53 at both Ser315 and Ser392 in vivo, suggesting the existence of two cooperating DNA damage-responsive pathways that play a role in stimulating p53-dependent gene expression. Our previous data has shown that cyclin A-cdk2 is the major enzyme responsible for modifying p53 at Ser315 in vivo after irradiation damage and in this report we dissect the mechanism of cyclinA-cdk2 binding to and phosphorylation of p53. Although cyclin B(1)-dependent protein kinases can phosphorylate small peptides containing the Ser315 site, cyclin A-cdk2 does not phosphorylate such small peptides suggesting that additional determinants are required for cyclin A-cdk2 interaction with p53. Peptide competition studies have localized a cyclin A interaction site to a Lys381Lys382Leu383Met384Phe385 sequence within C-terminal negative regulatory domain of human p53. An alanine mutation at any one of four key positions abrogates the efficacy of a synthetic peptide containing this motif as an inhibitor of cyclin A-cdk2 phosphorylation of p53 protein. Single amino acid mutations of full-length p53 protein at Lys382, Leu383, or Phe385 decreases cyclin A-cdk2 dependent phosphorylation at Ser315. Cyclin B(1)-cdk2 complexes are not inhibited by KKLMF motif-containing peptides nor is p53 phosphorylation by cyclin B-cdk2 reduced by mutation of the cyclin A interaction site. These data identifying a KKLMF cyclin A docking site on p53 protein highlight a common cyclin A interaction motif that is shared between the tumour suppressor proteins pRb and p53.

    Journal of molecular biology 2000;300;3;503-18

  • Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth.

    Zukerberg LR, Patrick GN, Nikolic M, Humbert S, Wu CL, Lanier LM, Gertler FB, Vidal M, Van Etten RA and Tsai LH

    Howard Hughes Medical Institute, Department of Pathology, Massachusetts General Hospital, Boston 02114, USA.

    Cyclin-dependent kinase 5 (Cdk5) is a small serine/threonine kinase that plays a pivotal role during development of the CNS. Cables, a novel protein, interacts with Cdk5 in brain lysates. Cables also binds to and is a substrate of the c-Abl tyrosine kinase. Active c-Abl kinase leads to Cdk5 tyrosine phosphorylation, and this phosphorylation is enhanced by Cables. Phosphorylation of Cdk5 by c-Abl occurs on tyrosine 15 (Y15), which is stimulatory for p35/Cdk5 kinase activity. Expression of antisense Cables in primary cortical neurons inhibited neurite outgrowth. Furthermore, expression of active Abl resulted in lengthening of neurites. The data provide evidence for a Cables-mediated interplay between the Cdk5 and c-Abl signaling pathways in the developing nervous system.

    Funded by: NINDS NIH HHS: R01-NS37007

    Neuron 2000;26;3;633-46

  • Cdk5 and MAPK are associated with complexes of cytoskeletal proteins in rat brain.

    Veeranna, Shetty KT, Takahashi M, Grant P, Pant HC and Veeranna GJ

    Laboratory of Neurochemistry, National Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bldg. 36, Rm. 4D20, Bethesda, MD 20892-4130, USA.

    Neurofilament proteins, the major cytoskeletal components of large myelinated axons, are highly phosphorylated by second messenger-dependent and -independent kinases. These kinases, together with tubulins and other cytoskeletal proteins, have been shown to bind to neurofilament preparations. Cdk5 and Erk2, proline-directed kinases in neuronal tissues, phosphorylate the Lys-Ser-Pro (KSP) repeats in tail domains of NF-H, NF-M, and other axonal proteins such as tau and synapsin. In neurofilament and microtubule preparations from rat brain, we demonstrated by Western blot analysis that cdk5, a neuronal cyclin dependent kinase and Erk1/2 were associated with complexes of NF proteins, tubulins and tau. Using P13(suc1) affinity chromatography, a procedure known to bind cdc2-like kinases in proliferating cells with high affinity, we obtained a P13 complex from a rat brain extract exhibiting the same profiles of cdk5 and Erk2 bound to cytoskeletal proteins. The phosphorylation activities of these preparations and the effect of the cdk5 inhibitor, butyrolactone, were consistent with the presence of active kinases. Finally, during a column fractionation and purification of Erk kinases from rat brain extracts, fractions enriched in Erk kinase activity also exhibited co-elution of phosphorylated NF-H, tubulin, tau and cdk5. We suggest that in mammalian brain, different kinases, their regulators and phosphatases form multimeric complexes with cytoskeletal proteins and regulate multisite phosphorylation from synthesis in the cell body to transport and assembly in the axon.

    Brain research. Molecular brain research 2000;76;2;229-36

  • p39 activates cdk5 in neurons, and is associated with the actin cytoskeleton.

    Humbert S, Dhavan R and Tsai L

    Howard Hughes Medical Institute, Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Cyclin-dependent kinase 5 (cdk5) is a small serine/threonine kinase that displays close sequence homology to the mitotically active cyclin-dependent kinases. Cdk5 has been shown to play an essential role in the development of the nervous system, including neuronal migration and neurite outgrowth. Cdk5 activation requires the presence of a regulatory activator such as p35. cdk5 -/- mice have much more extensive defects in the development of the nervous system than p35 -/- mice, leading to the speculation that other regulatory activators of cdk5 exist. Indeed, p39 is a p35 related protein isolated by sequence homology to p35. We show here that p39 associates with cdk5 in brain lysates, and that this complex is active in phosphorylation of histone H1. By extensive characterization of p39 subcellular localization in different cell types, we demonstrate the presence of p39 in lamellipodial and fillopodial structures of cells and in growth cones of neurons. We show that p39 colocalizes with actin, and cofractionates with the detergent insoluble cytoskeleton from brain. Further, p39 coimmunoprecipitates with actin in brain lysates. Finally, disruption of the actin cytoskeleton alters p39 subcellular localization as well as kinase activity of the p39/cdk5 complex. Therefore, our results reveal the existence of the p39/cdk5 complex in vivo and suggest that it might play a role in regulating actin cytoskeletal dynamics in cells.

    Funded by: NIGMS NIH HHS: GM53049

    Journal of cell science 2000;113 ( Pt 6);975-83

  • Cloning of three novel neuronal Cdk5 activator binding proteins.

    Ching YP, Qi Z and Wang JH

    Department of Biochemistry, Hong Kong University of Science and Technology, Kowloon. bcyching@ust.hk

    Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics. The active kinase consists of a catalytic subunit, Cdk5, and a 25 kDa activator protein (p25nck5a) derived from a 35 kDa neuronal-specific protein (p35nck5a). As an extension of our previous study (Qi, Z., Tang, D., Zhu, X., Fujita, D.J., Wang, J.H., 1998. Association of neurofilament proteins with neuronal Cdk5 activator. J. Biol. Chem. 270, 2329-2335), which showed that neurofilament is one of the p35nck5a-associated proteins, we now report the isolation of three other novel p35nck5a-associated proteins using the yeast two-hybrid screen. The full-length forms of these three novel proteins, designated C42, C48 and C53, have a molecular mass of 66, 24, and 57 kDa, respectively. Northern analysis indicates that these novel proteins are widely expressed in human tissues, including the heart, brain, skeletal muscle, placenta, lung, liver, kidney and pancreas. The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35nck5a complexed with Cdk5 from insect cell lysate. Among these three proteins, only C48 and C53 can be phosphorylated by Nclk, suggesting that they may be the substrates of Nclk. Sequence homology searches have suggested that the C48 protein is marginally related to restin protein, whereas the C42 protein has homologues of unknown function in Caenorhabditis elegans and Arabidopsis thaliana.

    Gene 2000;242;1-2;285-94

  • Phosphorylation of DARPP-32 by Cdk5 modulates dopamine signalling in neurons.

    Bibb JA, Snyder GL, Nishi A, Yan Z, Meijer L, Fienberg AA, Tsai LH, Kwon YT, Girault JA, Czernik AJ, Huganir RL, Hemmings HC, Nairn AC and Greengard P

    Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021, USA.

    The physiological state of the cell is controlled by signal transduction mechanisms which regulate the balance between protein kinase and protein phosphatase activities. Here we report that a single protein can, depending on which particular amino-acid residue is phosphorylated, function either as a kinase or phosphatase inhibitor. DARPP-32 (dopamine and cyclic AMP-regulated phospho-protein, relative molecular mass 32,000) is converted into an inhibitor of protein phosphatase 1 when it is phosphorylated by protein kinase A (PKA) at threonine 34. We find that DARPP-32 is converted into an inhibitor of PKA when phosphorylated at threonine 75 by cyclin-dependent kinase 5 (Cdk5). Cdk5 phosphorylates DARPP-32 in vitro and in intact brain cells. Phospho-Thr 75 DARPP-32 inhibits PKA in vitro by a competitive mechanism. Decreasing phospho-Thr 75 DARPP-32 in striatal slices, either by a Cdk5-specific inhibitor or by using genetically altered mice, results in increased dopamine-induced phosphorylation of PKA substrates and augmented peak voltage-gated calcium currents. Thus DARPP-32 is a bifunctional signal transduction molecule which, by distinct mechanisms, controls a serine/threonine kinase and a serine/threonine phosphatase.

    Funded by: NIDA NIH HHS: P01 DA010044

    Nature 1999;402;6762;669-71

  • Neuron-specific Cdk5 kinase is responsible for mitosis-independent phosphorylation of c-Src at Ser75 in human Y79 retinoblastoma cells.

    Kato G and Maeda S

    Department of Biochemistry, Yamanashi Medical University, Nakakoma, Yamanashi, 409-3898, Japan. gkato@res.yamanashi-med.ac.jp

    c-Src is phosphorylated at specific serine and threonine residues during mitosis in fibroblastic and epithelial cells. These sites are phosphorylated in vitro by the mitotic kinase Cdk1 (p34(cdc2)). In contrast, c-Src in Y79 human retinoblastoma cells, which are of neuronal origin, is phosphorylated at one of the mitotic sites, Ser75, throughout the cell cycle. The identity of the serine kinase that nonmitotically phosphorylates c-Src on Ser75 remains unknown. We now are able to show for the first time that Cdk5 kinase, which has the same consensus sequence as the Cdk1 and Cdk2 kinases, is required for the phosphorylation in asynchronous Y79 cells. The Ser75 phosphorylation was inhibited in a dose-dependent manner by butyrolactone I, a specific inhibitor of Cdk5-type kinases. Three stable subclones that have almost no kinase activity were selected by transfection of an antisense Cdk5-specific activator p35 construct into Y79 cells. The loss of the kinase activity caused an approximately 85% inhibition of the Ser75 phosphorylation. These results present compelling evidence that Cdk5/p35 kinase is responsible for the novel phosphorylation of c-Src at Ser75 in neuronal cells, raising the intriguing possibility that c-Src acts as an effector of Cdk5/p35 kinase during neuronal development.

    Journal of biochemistry 1999;126;5;957-61

  • Regulation of cyclin-dependent kinase 5 catalytic activity by phosphorylation.

    Sharma P, Sharma M, Amin ND, Albers RW and Pant HC

    Laboratory of Neurochemistry, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.

    Cyclin-dependent kinase 5 (cdk5) is found in an active form only in neuronal cells. Activation by virtue of association with the cyclin-like neuronal proteins p35 (or its truncated form p25) and p39 is the only mechanism currently shown to regulate cdk5 catalytic activity. In addition to cyclin binding, other members of the cdk family require for maximal activation phosphorylation of a Ser/Thr residue (Thr(160) in the case of cdk-2) that is conserved in all cdks except cdk8. This site is phosphorylated by cdk-activating kinases, which, however, do not phosphorylate cdk5. To examine the possible existence of a phosphorylation-dependent regulatory mechanism in the case of cdk5, we have metabolically labeled PC12 cells with (32)P(i) and shown that the endogenous cdk5 is phosphorylated. Bacterially expressed cdk5 also can be phosphorylated by PC12 cell lysates. Phosphorylation of cdk5 by a PC12 cell lysate results in a significant increase in cdk5/p25 catalytic activity. Ser(159) in cdk5 is homologous to the regulatory Thr(160) in cdk2. A Ser(159)-to-Ala (S159A) cdk5 mutant did not show similar activation, which suggests that cdk5 is also regulated by phosphorylation at this site. Like other members of the cdk family, cdk5 catalytic activity is influenced by both p25 binding and phosphorylation. We show that the cdk5-activating kinase (cdk5AK) is distinct from the cdk-activating kinase (cyclin H/cdk7) that was reported previously to neither phosphorylate cdk5 nor affect its activity. We also show that casein kinase I, but not casein kinase II, can phosphorylate and activate cdk5 in vitro.

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;20;11156-60

  • New phosphorylation sites identified in hyperphosphorylated tau (paired helical filament-tau) from Alzheimer's disease brain using nanoelectrospray mass spectrometry.

    Hanger DP, Betts JC, Loviny TL, Blackstock WP and Anderton BH

    Department of Neuroscience, Institute of Psychiatry, London, England, UK.

    Paired helical filaments (PHFs) are the structural constituents of neurofibrillary tangles in Alzheimer's disease and are composed of hyperphosphorylated forms of the microtubule-associated protein tau (PHF-tau). Pathological hyperphosphorylation of tau is believed to be an important contributor to the destabilisation of microtubules and their subsequent disappearance from tangle-bearing neurons in Alzheimer's disease, making elucidation of the mechanisms that regulate tau phosphorylation an important research goal. Thus, it is essential to identify, preferably by direct sequencing, all of the sites in PHF-tau that are phosphorylated, a task that is incomplete because of the difficulty to date of purifying insoluble PHF-tau to homogeneity and in sufficient quantities for structural analysis. Here we describe the solubilisation of PHF-tau followed by its purification by Mono Q chromatography and reversed-phase HPLC. Phosphopeptides from proteolytically digested PHF-tau were sequenced by nanoelectrospray mass spectrometry. We identified 22 phosphorylation sites in PHF-tau, including five sites not previously identified. The combination of our new data with previous reports shows that PHF-tau can be phosphorylated on at least 25 different sites.

    Funded by: Wellcome Trust

    Journal of neurochemistry 1998;71;6;2465-76

  • Tau is phosphorylated by GSK-3 at several sites found in Alzheimer disease and its biological activity markedly inhibited only after it is prephosphorylated by A-kinase.

    Wang JZ, Wu Q, Smith A, Grundke-Iqbal I and Iqbal K

    Chemical Neuropathology Department, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314-6399, USA.

    Alzheimer disease is characterized by a specific type of neuronal degeneration in which the microtubule associated protein tau is abnormally hyperphosphorylated causing the disruption of the microtubule network. We have found that the phosphorylation of human tau (tau3L) by A-kinase, GSK-3 or CK-1 inhibits its microtubule assembly-promoting and microtubule-binding activities. However, the inhibition of these activities of tau by GSK-3 is significantly increased if tau is prephosphorylated by A-kinase or CK-1. The most potent inhibition is observed by combination phosphorylation of tau with A-kinase and GSK-3. Under these conditions, only very few microtubules are seen by electron microscopy. Sequencing of 32P-labeled trypsin phosphopeptides from tau prephosphorylated by A-kinase (using unlabeled ATP) and further phosphorylated by GSK-3 in the presence of [gamma-32P]ATP revealed that Ser-195, Ser-198, Ser-199, Ser-202, Thr-205, Thr-231, Ser-235, Ser-262, Ser-356 and Ser-404 are phosphorylated, whereas if tau is not prephosphorylated by A-kinase, GSK-3 phosphorylates it at Thr-181, Ser-184, Ser-262, Ser-356 and Ser-400. These data suggest that (i) prephosphorylation of tau by A-kinase makes additional and different sites accessible for phosphorylation by GSK-3; (ii) phosphorylation of tau at these additional sites further inhibits the biological activity of tau in its ability to bind to microtubules and promote microtubule assembly. Thus a combined role of A-kinase and GSK-3 should be considered in Alzheimer neurofibrillary degeneration.

    Funded by: NIA NIH HHS: AG05892, AG08076; NINDS NIH HHS: NS18105; ...

    FEBS letters 1998;436;1;28-34

  • Phosphorylation of tau at both Thr 231 and Ser 262 is required for maximal inhibition of its binding to microtubules.

    Sengupta A, Kabat J, Novak M, Wu Q, Grundke-Iqbal I and Iqbal K

    New York State Institute for Basic Research in Developmental Disabilities, Staten Island 10314, USA. kiqbal@admin.con2.com

    The paired helical filaments (PHFs) found in Alzheimer's disease (AD) brains are composed primarily of the microtubule-associated protein tau. PHF-tau is in a hyperphosphorylated state and is unable to promote microtubule assembly. We investigated whether the inhibition of tau binding to microtubules is increased when tau is phosphorylated by different kinases in combination with GSK-3. We found that when tau was first phosphorylated by A-kinase, C-kinase, cdk5, or CaM kinase II and then by GSK-3, its binding to microtubules was inhibited by 45, 61, 78, and 79%, respectively. Further, the kinase combinations cdk5/GSK-3 and CaM kinase II/GSK-3 rapidly phosphorylated the sites Thr 231 and Ser 235. When these sites were individually replaced by Ala and the phosphorylation experiments repeated, tau binding to microtubules was inhibited by 54 and 71%, respectively. By comparison, when Ser 262 was replaced by Ala, tau binding to microtubules was inhibited by only 8% after phosphorylation by CaM kinase II. From these observations we estimate that the phosphorylation of Thr 231, Ser 235, and Ser 262 contributes approximately 26, approximately 9, and approximately 33%, respectively, of the overall inhibition of tau binding to microtubules. Together, our results indicate that the binding of tau to microtubules is controlled by the phosphorylation of several sites, among which are Thr 231, Ser 235, and Ser 262.

    Funded by: NIA NIH HHS: AG05892, AG08076; NINDS NIH HHS: NS18105

    Archives of biochemistry and biophysics 1998;357;2;299-309

  • The endogenous and cell cycle-dependent phosphorylation of tau protein in living cells: implications for Alzheimer's disease.

    Illenberger S, Zheng-Fischhöfer Q, Preuss U, Stamer K, Baumann K, Trinczek B, Biernat J, Godemann R, Mandelkow EM and Mandelkow E

    Max-Planck-Unit for Structural Molecular Biology, D-22603 Hamburg, Germany.

    In Alzheimer's disease the neuronal microtubule-associated protein tau becomes highly phosphorylated, loses its binding properties, and aggregates into paired helical filaments. There is increasing evidence that the events leading to this hyperphosphorylation are related to mitotic mechanisms. Hence, we have analyzed the physiological phosphorylation of endogenous tau protein in metabolically labeled human neuroblastoma cells and in Chinese hamster ovary cells stably transfected with tau. In nonsynchronized cultures the phosphorylation pattern was remarkably similar in both cell lines, suggesting a similar balance of kinases and phosphatases with respect to tau. Using phosphopeptide mapping and sequencing we identified 17 phosphorylation sites comprising 80-90% of the total phosphate incorporated. Most of these are in SP or TP motifs, except S214 and S262. Since phosphorylation of microtubule-associated proteins increases during mitosis, concomitant with increased microtubule dynamics, we analyzed cells mitotically arrested with nocodazole. This revealed that S214 is a prominent phosphorylation site in metaphase, but not in interphase. Phosphorylation of this residue strongly decreases the tau-microtubule interaction in vitro, suppresses microtubule assembly, and may be a key factor in the observed detachment of tau from microtubules during mitosis. Since S214 is also phosphorylated in Alzheimer's disease tau, our results support the view that reactivation of the cell cycle machinery is involved in tau hyperphosphorylation.

    Molecular biology of the cell 1998;9;6;1495-512

  • Characterization of tau phosphorylation in glycogen synthase kinase-3beta and cyclin dependent kinase-5 activator (p23) transfected cells.

    Michel G, Mercken M, Murayama M, Noguchi, Ishiguro K, Imahori K and Takashima A

    Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan.

    One of the histopathological markers in Alzheimer's disease is the accumulation of hyperphosphorylated tau in neurons ca 875 lled neurofibrillary tangles (NFT) composing paired helical filaments (PHF). Combined tau protein kinase II (TPK II), which consists of CDK5 and its activator (p23), and glycogen synthase kinase-3beta (GSK-3beta) phosphorylate tau to the PHF-form in vitro. To investigate tau phosphorylation by these kinases in intact cells, the phosphorylation sites were examined in detail using well-characterized phosphorylation-dependent anti-tau antibodies after overexpressing the kinases in COS-7 cells with a human tau isoform. The overexpression of tau in COS-7 cells showed extensive phosphorylation at Ser-202 and Ser-404. The p23 overexpression induced a mobility shift of tau, but most of the phosphorylation sites overlapped the endogenous phosphorylation sites. GSK-3beta transfection showed the phosphorylation at Ser-199, Thr-231, Ser-396, and Ser-413. Triplicated transfection resulted in phosphorylation of tau at 8 observed sites (Ser-199, Ser-202, Thr-205, Thr-231, Ser-235, Ser-396, Ser-404, and Ser-413).

    Biochimica et biophysica acta 1998;1380;2;177-82

  • Cyclin-dependent kinase-5 is associated with lipofuscin in motor neurones in amyotrophic lateral sclerosis.

    Bajaj NP, Al-Sarraj ST, Anderson V, Kibble M, Leigh N and Miller CC

    Department of Clinical Neurosciences, The Institute of Psychiatry, London, UK. spgtnpb@iop.bpmf.ac.uk

    We have studied the distribution of cyclin dependent kinase-5 (cdk-5) within spinal cord in sporadic and two superoxide dismutase type 1 (SOD1) familial cases of amyotrophic lateral sclerosis (ALS). Although most neurofilament accumulations in ALS motor neurones did not appear to contain high levels of cdk-5, intense cdk-5 immunoreactivity was observed in perikarya of degenerating neurones in many ALS cases. Here, cdk-5 co-localised with lipofuscin. Co-localisation of cdk-5 with lipofuscin was also observed in some aged non-affected controls although this labelling was less intense than the ALS cases. The biogenesis of lipofuscin is believed to be linked to oxidative stress and oxidative stress and free radical damage have been suggested to be part of the pathogenic process of ALS, possibly involving apoptotic mechanisms. cdk-5 has recently been associated with apoptosis. These observations suggest a role for cdk-5 in the pathogenesis of ALS.

    Funded by: Wellcome Trust

    Neuroscience letters 1998;245;1;45-8

  • Sequential phosphorylation of Tau by glycogen synthase kinase-3beta and protein kinase A at Thr212 and Ser214 generates the Alzheimer-specific epitope of antibody AT100 and requires a paired-helical-filament-like conformation.

    Zheng-Fischhöfer Q, Biernat J, Mandelkow EM, Illenberger S, Godemann R and Mandelkow E

    Max-Planck-Unit for Structural Molecular Biology, Hamburg, Germany.

    AT100 is a monoclonal antibody highly specific for phosphorylated Tau in Alzheimer paired helical filaments. Here we show that the epitope is generated by a complex sequence of sequential phosphorylation, first of Ser199, Ser202 and Thr205 (around the epitope of antibody AT8), next of Thr212 by glycogen synthase kinase (GSK)-3beta (a proline-directed kinase), then of Ser214 by protein kinase A (PKA). Conversely, if Ser214 is phosphorylated first it protects Thr212 and the Ser-Pro motifs around the AT8 site against phosphorylation, and the AT100 epitope is not formed. The generation of the AT100 epitope requires a conformation of tau induced by polyanions such as heparin, RNA or poly(Glu), conditions which also favor the formation of paired helical filaments. The Alzheimer-like phosphorylation can be induced by brain extracts. In the extract, the kinases responsible for generating the AT100 epitope are GSK-3beta and PKA, which can be inhibited by their specific inhibitors LiCl and RII, respectively. A cellular model displaying the reaction with AT100 is presented by Sf9 insect cells transfected with Tau. Knowledge of the events and kinases generating the AT100 epitope in cells might allow us to study the degeneration of the cytoskeleton in Alzheimer's disease.

    European journal of biochemistry 1998;252;3;542-52

  • Regulation of Munc-18/syntaxin 1A interaction by cyclin-dependent kinase 5 in nerve endings.

    Shuang R, Zhang L, Fletcher A, Groblewski GE, Pevsner J and Stuenkel EL

    Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109-0622, USA.

    The Munc-18-syntaxin 1A complex has been postulated to act as a negative control on the regulated exocytotic process because its formation blocks the interaction of syntaxin with vesicle SNARE proteins. However, the formation of this complex is simultaneously essential for the final stages of secretion as evidenced by the necessity of Munc-18's homologues in Saccharomyces cerevisiae (Sec1p), Drosophila (ROP), and Caenorhabditis elegans (Unc-18) for proper secretion in these organisms. As such, any event that regulates the interaction of these two proteins is important for the control of secretion. One candidate for such regulation is cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division cycle kinases that has recently been copurified with Munc-18 from rat brain. The present study shows that Cdk5 bound to its neural specific activator p35 not only binds to Munc-18 but utilizes it as a substrate for phosphorylation. Furthermore, it is demonstrated that Munc-18 that has been phosphorylated by Cdk5 has a significantly reduced affinity for syntaxin 1A. Finally, it is shown that Cdk5 can also bind to syntaxin 1A and that a complex of Cdk5, p35, Munc-18, and syntaxin 1A can be fashioned in the absence of ATP and promptly disassembled upon the addition of ATP. These results suggest a model in which p35-activated Cdk5 becomes localized to the Munc-18-syntaxin 1A complex by its affinity for both proteins so that it may phosphorylate Munc-18 and thus permit the positive interaction of syntaxin 1A with upstream protein effectors of the secretory mechanism.

    Funded by: NINDS NIH HHS: NS31888, NS36227

    The Journal of biological chemistry 1998;273;9;4957-66

  • Cyclin D2 interacts with cdk-5 and modulates cellular cdk-5/p35 activity.

    Guidato S, McLoughlin DM, Grierson AJ and Miller CC

    Department of Clinical Neurosciences, Institute of Psychiatry, London, England.

    Cyclin-dependent kinase-5 (cdk-5) is a serine/threonine kinase that displays neurone-specific activity. Experimental manipulation of cdk-5 expression in neurones has shown that cdk-5 is essential for proper development of the nervous system and, in particular, for outgrowth of neurites. Such observations suggest that cdk-5 activity must be tightly controlled during development of the nervous system. To identify possible regulators of cdk-5, we used the yeast two-hybrid system to search for proteins that interact with cdk-5. In two independent yeast transformation events, cyclin D2 interacted with cdk-5. Immunoprecipitation experiments confirmed that cyclin D2 and cdk-5 interact in mammalian cells. Cyclin D2 did not activate cdk-5 as assayed using three different substrates, which was in contrast to a known cdk-5 activator, p35. However, cyclin D2 expression led to a decrease in cdk-5/p35 activity in transfected cells. As cyclin D2 and cdk-5 are known to share overlapping patterns of expression during development of the CNS, the results presented here suggest a role for cyclin D2 in modulating cdk-5 activity in postmitotic developing neurones.

    Funded by: Wellcome Trust

    Journal of neurochemistry 1998;70;1;335-40

  • Identification of DNA replication and cell cycle proteins that interact with PCNA.

    Loor G, Zhang SJ, Zhang P, Toomey NL and Lee MY

    University of Miami, Department of Biochemistry and Molecular Biology and Medicine, Miami, FL 33101, USA.

    The identity of DNA replication proteins and cell cycle regulatory proteins which can be found in complexes involving PCNA were investigated by the use of PCNA immobilized on Sepharose 4B. A column containing bovine serum albumin (BSA) bound to Sepharose was used as a control. Fetal calf thymus extracts were chromatographed on PCNA-Sepharose and BSA-Sepharose. The columns were washed and then eluted with 0.5 M KCl. The salt eluates were examined for the presence of both DNA replication proteins (Pol alpha, delta, straightepsilon, PCNA, RFC, RFA, DNA ligase I, NDH II, Topo I and Topo II) and cell cycle proteins (Cyclins A, B1, D1, D2, D3, E, CDK2, CDK4, CDK5 and p21) by western blotting with specific antibodies. The DNA replication proteins which bound to PCNA-Sepharose included DNA polymerase delta and straightepsilon, PCNA, the 37 and 40 kDa subunits of RFC, the 70 kDa subunit of RPA, NDH II and topoisomerase I. No evidence for the binding of DNA polymerase alpha, DNA ligase I or topoisomerase II was obtained. Of the cell cycle proteins investigated, CDK2, CDK4 and CDK5 were bound. This study presents strong evidence that PCNA is a component of protein complexes containing DNA replication, repair and cell cycle regulatory proteins.

    Funded by: NIGMS NIH HHS: GM31973

    Nucleic acids research 1997;25;24;5041-6

  • Phosphorylation by neuronal cdc2-like protein kinase promotes dimerization of Tau protein in vitro.

    Paudel HK

    Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital and Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, H3T 1E2, Canada. MDHP@MUSICA.McGILL.CA

    In Alzheimer's disease, the microtubule-associated protein tau forms paired helical filaments (PHFs) that are the major structural component of neurofibrillary tangles. Although tau isolated from PHFs (PHF-tau) is abnormally phosphorylated, the role of this abnormal phosphorylation in PHF assembly is not known. Previously, neuronal cdc2-like protein kinase (NCLK) was shown to phosphorylate tau on sites that are abnormally phosphorylated in PHF-tau (Paudel, H. K., Lew, J., Ali, Z., and Wang, J. H. (1993) J. Biol. Chem. 268, 23512-23518). In this study, phosphorylation by NCLK was found to promote dimerization of recombinant human tau (R-tau) and brain tau (B-tau) purified from brain extract. Chemical cross-linking by disuccinimidyl suberate (DSS), a homobifunctional chemical cross-linker that specifically cross-linked R-tau dimers, and a Superose 12 gel filtration chromatography revealed that R-tau preparations contain mixtures of monomeric and dimeric R-tau species. When the structure of NCLK-phosphorylated R-tau was studied by a similar approach, DSS preferentially cross-linked the phosphorylated R-tau over the nonphosphorylated R-tau, and the phosphorylated R-tau eluted as a dimeric species from the gel filtration column. Phosphorylated R-tau became resistant to DSS upon dephosphorylation and was recovered as a monomeric species from the gel filtration column. In the presence of a low concentration of dithiothreitol (1.65 microM), R-tau formed disulfide cross-linked R-tau dimers. When compared, phosphorylated R-tau formed more disulfide cross-linked dimers than the nonphosphorylated R-tau. B-tau also was specifically cross-linked to dimers by DSS. When B-tau and NCLK-phosphorylated B-tau were treated with DSS, phosphorylated B-tau was preferentially cross-linked over nonphosphorylated counterpart. Taken together, these results suggest that phosphorylation by NCLK promotes dimerization and formation of disulfide cross-linked tau dimers, which is suggested to be the key step leading to PHF assembly (Schweers, O., Mandelkow, E.-M., Biernat, J., and Mandelkow, E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8463-8467).

    The Journal of biological chemistry 1997;272;45;28328-34

  • BRCA1 proteins are transported to the nucleus in the absence of serum and splice variants BRCA1a, BRCA1b are tyrosine phosphoproteins that associate with E2F, cyclins and cyclin dependent kinases.

    Wang H, Shao N, Ding QM, Cui J, Reddy ES and Rao VN

    Department of Human Genetics, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19102, USA.

    BRCA1, a familial breast and ovarian cancer susceptibility gene encodes nuclear phosphoproteins that function as tumor suppressors in human breast cancer cells. Previously, we have shown that overexpression of a BRCA1 splice variant BRCA1a accelerates apoptosis in human breast cancer cells. In an attempt to determine whether the subcellular localization of BRCA1 is cell cycle regulated, we have studied the subcellular distribution of BRCA1 in asynchronous and growth arrested normal, breast and ovarian cancer cells using different BRCA1 antibodies by immunofluorescence and immunohistochemical staining. Upon serum starvation of NIH3T3, some breast and ovarian cancer cells, most of the BRCA1 protein redistributed to the nucleus revealing a new type of regulation that may modulate the activity of BRCA1 gene. We have also characterized two new variant BRCA1 proteins (BRCA1a/p110 and BRCA1b/ p100) which are phosphoproteins containing phosphotyrosine. Immunofluorescence and Western blotting analysis indicate cytoplasmic and nuclear localization of BRCA1a and BRCA1b proteins. To elucidate the biological function of BRCA1, we created a bacterial fusion protein of glutathione-transferase (GST) and BRCA1 zinc finger domain and detected two cellular proteins with molecular weights of approximately 32 and 65 kD, one of which contains phosphotyrosine designated p32 and p65 BRCA1 interacting proteins (BIP) that specifically interact with BRCA1. Western blot analysis of BIP with cyclins/CDKs and E2F antisera indicated association with cdc2, cdk2, cdk4, cyclin B, cyclin D, cyclin A and E2F-4 but not with cdk3, cdk5, cdk6, E2F-1, E2F-2, E2F-3, E2F-5 and cyclin E. Furthermore, we have also demonstrated a direct interaction of in vitro translated BRCA1a and BRCA1b proteins with recombinant cyclin A, cyclin B1, cyclin D1, cdc2, cdk2 and E2F fusion proteins in vitro. Taken together these results seem to suggest that BRCA1 could be an important negative regulator of cell cycle that functions through interaction with E2F transcriptional factors and phosphorylation by cyclins/cdk complexes with the zinc ring finger functioning as a major protein-protein interaction domain. If the interactions we observe in vitro is also seen in vivo then it may be possible that lack or impaired binding of the disrupted BRCA1 proteins to E2F, cyclins/CDKs in patients with mutations in the zinc finger domain could deprive the cell of an important mechanism for braking cell proliferation leading to the development of breast and ovarian cancers.

    Funded by: NCI NIH HHS: CA 50507, CA 51083, CA 57322; ...

    Oncogene 1997;15;2;143-57

  • Phosphorylation of microtubule-associated protein tau by stress-activated protein kinases.

    Goedert M, Hasegawa M, Jakes R, Lawler S, Cuenda A and Cohen P

    MRC Laboratory of Molecular Biology, Cambridge, UK.

    The paired helical filament, which comprises the major fibrous element of the neurofibrillary lesions of Alzheimer's disease, is composed of hyperphosphorylated microtubule-associated protein tau. Many of the hyperphosphorylated sites in tau are serine/threonine-prolines. Here we show that the stress-activated protein (SAP) kinases SAPK1gamma (also called JNK1), SAPK2a (also called p38, RK, CSBPs, Mpk2 and Mxi2), SAPK2b (also called p38beta), SAPK3 (also called ERK6 and p38gamma) and SAPK4 phosphorylate tau at many serine/threonine-prolines, as assessed by the generation of the epitopes of phosphorylation-dependent anti-tau antibodies. Based on initial rates of phosphorylation, tau was found to be a good substrate for SAPK4 and SAPK3, a reasonable substrate for SAPK2b and a relatively poor substrate for SAPK2a and SAPK1gamma. Phosphorylation of tau by SAPK3 and SAPK4 resulted in a marked reduction in its ability to promote microtubule assembly. These findings double the number of candidate protein kinases for the hyperphosphorylation of tau in Alzheimer's disease and other neurodegenerative disorders.

    FEBS letters 1997;409;1;57-62

  • Interaction between Cdc37 and Cdk4 in human cells.

    Lamphere L, Fiore F, Xu X, Brizuela L, Keezer S, Sardet C, Draetta GF and Gyuris J

    Mitotix, Inc., Cambridge, Massachusetts 02139, USA.

    Using the yeast two-hybrid system we have identified novel potential Cdk4 interacting proteins. Here we described the interaction of Cdk4 with a human homologue of the yeast Drosophila CDC37 gene products. Cdc37 protein specifically interacts with Cdk4 and Cdk6, but not with Cdc2, Cdk2, Cdk3, Cdk5 and any of a number of cyclins tested. Cdc37 is not an inhibitor nor an activator of the Cdk4/cyclin D1 kinase, while it appears to facilitate complex assembly between Cdk4, and cyclin D1 in vitro. Cdc37 competes with p16 for binding to Cdk4, suggesting that p16 might exert part of its inhibitory function by affecting the formation of Cdk4/cyclin D1 complexes via Cdc37.

    Oncogene 1997;14;16;1999-2004

  • Identification of functional domains in the neuronal Cdk5 activator protein.

    Poon RY, Lew J and Hunter T

    Salk Institute for Biological Studies, La Jolla, California 92037, USA.

    Cyclin-dependent kinase 5 (Cdk5) is activated by the neuronal-specific activator protein, p35. In contrast to the activation of typical CDKs by cyclin subunits, p35.Cdk5 was not further activated by the CDK-activating kinase (CAK) and was neither phosphorylated nor inhibited by the Tyr-15-specific Wee1 kinase. The previously identified proteolytic active fragment of p35, p25 (residues 91-307) as well as the slightly smaller fragment containing residues 109-291, was found to be sufficient to bind and activate Cdk5. Other CDKs, including Cdk2, associated weakly with p25. However, their kinase activity was only activated to the low level observed for cyclin A.Cdk2 without Thr-160 phosphorylation, and phosphorylation of Thr-160 in Cdk2 did not activate the p25.Cdk2 complex further. We have identified distinct regions in p35 required for binding to Cdk5 or activation of Cdk5. Residues approximately 150-200 of p35 were sufficient for binding to Cdk5, but residues approximately 279-291 were needed in addition for activation of Cdk5 in vitro.

    Funded by: NCI NIH HHS: CA14195, CA39780

    The Journal of biological chemistry 1997;272;9;5703-8

  • GAK: a cyclin G associated kinase contains a tensin/auxilin-like domain.

    Kanaoka Y, Kimura SH, Okazaki I, Ikeda M and Nojima H

    Department of Molecular Genetics, Osaka University, Yamadaoka, Suita, Japan.

    We have cloned a cDNA encoding a novel association partner of cyclin G by West-Western blotting. The cDNA encodes a protein that harbors a Ser/Thr protein kinase-like catalytic domain at the N-terminal. Hence, we named it GAK (cyclin G-associated kinase). The long C-terminal extension shares homology with tensin and auxilin, and contains a leucine zipper region. Co-immunoprecipitation and Western blotting showed that GAK and cyclin G associate together in vivo. GAK also co-precipitated with CDK5, and CDK5 was found to be associated with cyclin G. We also showed by BIAcore analysis that the GAK-cyclin G interaction was direct.

    FEBS letters 1997;402;1;73-80

  • Intracellular localization of cyclin-dependent kinase 5 (CDK5) in mouse neuron: CDK5 is located in both nucleus and cytoplasm.

    Ino H and Chiba T

    Third Department of Anatomy, Chiba University School of Medicine, Japan.

    Cyclin-dependent kinase 5 (CDK5) is one of the cyclin-dependent kinases, and is expressed in mature neurons. CDK5 has been postulated to be a neurofilament or tau protein kinase, because it phosphorylates neurofilaments and tau protein (microtubule-associated protein tau) in vitro. It has been reported that CDK5 was immunohistochemically detected only in axons of neurons. We here report the immunohistochemical study of CDK5 using two distinct antibodies, one recognizing the N-terminal of CDK5 and the other the C-terminal. Immunoreactivity of CDK5 was found not only in axons, but also intensively in nuclei, though not in nucleoli, of neurons in the mouse central and peripheral nervous systems. The nuclear CDK5 possibly has a physiological function distinct from the neurofilament or tau protein kinase.

    Brain research 1996;732;1-2;179-85

  • Site-specific phosphorylation of synapsin I by mitogen-activated protein kinase and Cdk5 and its effects on physiological functions.

    Matsubara M, Kusubata M, Ishiguro K, Uchida T, Titani K and Taniguchi H

    Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-11, Japan.

    Posttranslational modifications of synapsin I, a major phosphoprotein in synaptic terminals, were studied by mass spectrometry. In addition to a well known phosphorylation site by calmodulin-dependent protein kinase II (CaM kinase II), a hitherto unrecognized site (Ser553) was found phosphorylated in vivo. The phosphorylation site is immediately followed by a proline, suggesting that the protein is an in vivo substrate of so-called proline-directed protein kinase(s). To identify the kinase involved, three proline-directed protein kinases expressed highly in the brain, i.e. mitogen-activated protein (MAP) kinase, Cdk5-p23, and glycogen synthase kinase 3beta, were tested for the in vitro phosphorylation of synapsin I. Only MAP kinase and Cdk5-p23 phosphorylated synapsin I stoichiometrically. The phosphorylation sites were determined to be Ser551 and Ser553 with Cdk5-p23, and Ser62, Ser67, and Ser551 with MAP kinase. Upon phosphorylation with MAP kinase, synapsin I showed reduced F-actin bundling activity, while no significant effect on the interaction was observed with the protein phosphorylated with Cdk5-p23. These results raise the possibility that the so-called proline-directed protein kinases together with CaM kinase II and cAMP-dependent protein kinase play an important role in the regulation of synapsin I function.

    The Journal of biological chemistry 1996;271;35;21108-13

  • In vitro phosphorylation of the cytoplasmic domain of the amyloid precursor protein by glycogen synthase kinase-3beta.

    Aplin AE, Gibb GM, Jacobsen JS, Gallo JM and Anderton BH

    Department of Neuroscience, Institute of Psychiatry, London, England.

    The two pathological lesions found in the brains of Alzheimer's disease patients, neurofibrillary tangles and neuritic plaques, are likely to be formed through a common pathway. Neurofibrillary tangles are intracellular aggregates of paired helical filaments, the main component of which is hyperphosphorylated forms of the microtubule-associated protein tau. Extracellular neuritic plaques and diffuse and vascular amyloid deposits are aggregates of beta-amyloid protein, a 4-kDa protein derived from the amyloid precursor protein (APP). Using conditions in vitro under which two proline-directed protein kinases, glycogen synthase kinase-3beta (GSK-3beta) and mitogen-activated protein kinase (MAPK), were able to hyperphosphorylate tau, GSK-3beta but not MAPK phosphorylated recombinant APPcyt. The sole site of phosphorylation in APPcyt by GSK-3beta was determined by phosphoamino acid analysis and phosphorylation of APPcyt mutant peptides to be Thr743 (numbering as for APP770). This site was confirmed by endoproteinase Glu-C digestion of APPcyt and peptide sequencing. The ability of GSK-3beta to phosphorylate APPcyt and tau provides a putative link between the two lesions and indicates a critical role of GSK-3beta in the pathogenesis of Alzheimer's disease.

    Funded by: Wellcome Trust

    Journal of neurochemistry 1996;67;2;699-707

  • Hyperphosphorylation of tau in PHF.

    Morishima-Kawashima M, Hasegawa M, Takio K, Suzuki M, Yoshida H, Watanabe A, Titani K and Ihara Y

    Department of Neuropathology, Faculty of Medicine, University of Tokyo, Japan.

    Tau in PHF is known to be highly phosphorylated and immunochemical study has indicated the similarity of the phosphorylation between PHF-tau and fetal tau. We have determined the exact phosphorylation sites in both PHF-tau and fetal rat tau by ion-spray mass spectrometry and sequencing of ethanethiol-modified peptides. In PHF-tau, 19 sites have been identified; all the phosphorylation sites except for Ser-262 are localized to the amino- and carboxyl-terminal flanking regions of the microtubule-binding domain. Half of them are shared by fetal tau. Thus, PHF-tau is much more phosphorylated. Whereas most of the sites in fetal tau are proline-directed, half of them in PHF-tau are nonproline-directed. Overall, the hyperphosphorylation of PHF-tau can be considered to consist of fetal-type phosphorylation and additional proline-directed and nonproline-directed phosphorylation. This extraphosphorylation may provide PHF-tau with the unusual characteristics including assembly incompetence.

    Neurobiology of aging 1995;16;3;365-71; discussion 371-80

  • Association of proliferating cell nuclear antigen with cyclin-dependent kinases and cyclins in normal and transformed human T lymphocytes.

    Szepesi A, Gelfand EW and Lucas JJ

    Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206.

    The proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and appears to be needed for both DNA synthesis and DNA repair. It is present in low amount in resting normal human T lymphocytes and, upon mitogenic stimulation with phorbol dibutyrate and ionomycin, begins to increase in mid-G1 phase, approximately 12 to 15 hours before entry into S phase. PCNA continues to increase in amount throughout the cell cycle and remains high in proliferating cultures. PCNA was extracted from activated normal T cells and from the transformed T-lymphoblastoid cell line Jurkat by a method that recovered approximately 98% of total cellular PCNA but yet retained its associations with other proteins. PCNA immunoprecipitates possessed H1 histone kinase activity, which increased in parallel with increasing cellular content of PCNA. Both the cdc2 and cdk2 kinases were found associated with PCNA in normal T cells, in amounts consistent with detected kinase activity. The results indicate that PCNA is not an inhibitory molecule of cdk/cyclin activity. Both normal and transformed T cells contained PCNA in association with cdk2, cdk4, cdk5, and cdk6, with the amount of PCNA associated with these molecules increasing in the order listed. Relatively high amounts of PCNA were also found associated with cyclins D2 and D3, the major cyclin partners of cdk6 in T cells. Though detected in normal cells, PCNA/cdc2 complexes were present in exceedingly low amount, if at all, in Jurkat cells. This cell line appeared to contain more of nearly all of the cdk and cyclin molecules analyzed, but there seemed to be little difference in the patterns of association of these molecules with PCNA in the cell line as compared with normal human T cells.

    Funded by: NIAID NIH HHS: AI-26490, AI-29704, AI-29903

    Blood 1994;84;10;3413-21

  • Expression of CDK5 (PSSALRE kinase), a neural cdc2-related protein kinase, in the mature and developing mouse central and peripheral nervous systems.

    Ino H, Ishizuka T, Chiba T and Tatibana M

    Department of Anatomy, Chiba University School of Medicine, Japan.

    CDK5 is a cdc2-related protein kinase that is known to be highly expressed in mature brain. In this study, we obtained a mouse CDK5 cDNA by screening an adult mouse cDNA library. Northern blot analysis demonstrated that the mouse CDK5 mRNA was expressed especially highly in brain, and moderately in kidney, testis and ovary. In brain the expression of CDK5 is already seen at embryonal 12.5 days (E12.5), and it gradually increases through the embryonal stage. After birth, the expression is maintained at a high level to adulthood. In situ hybridization demonstrated that the expression of CDK5 mRNA was distributed in neurons throughout the brain, spinal cord and peripheral ganglia, especially in the hippocampal pyramidal cells, cerebellar Purkinje cells, cortical neurons, olfactory mitral cells, mesencephalic and motor trigeminal nuclei and trigeminal ganglion. In any portion, no apparent expression was observed in glia. During development, the expression of CDK5 was already seen at E12.5 intensely in trigeminal and dorsal root ganglia, and moderately and diffusely in the central nervous system. The expression pattern of CDK5 is quite in contrast with that of CDC2. The fact that CDK5 is expressed in terminally differentiated non-dividing neurons predicts an alternative function(s) in addition to controlling the cell cycle.

    Brain research 1994;661;1-2;196-206

  • A brain-specific activator of cyclin-dependent kinase 5.

    Lew J, Huang QQ, Qi Z, Winkfein RJ, Aebersold R, Hunt T and Wang JH

    MRC Group in Signal Transduction, University of Calgary, Alberta, Canada.

    Phosphorylation of the neurofilament proteins of high and medium relative molecular mass, as well as of the Alzheimer's tau protein, is thought to be catalysed by a protein kinase with Cdc2-like substrate specificity. We have purified a novel Cdc2-like kinase from bovine brain capable of phosphorylating both the neurofilament proteins and tau. The purified enzyme is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a novel regulatory subunit, p25 (ref. 8). When overexpressed and purified from Escherichia coli, p25 can activate Cdk5 in vitro. Unlike Cdk5, which is ubiquitously expressed in human tissue, the p25 transcript is expressed only in brain. A full-length complementary DNA clone showed that p25 is a truncated form of a larger protein precursor, p35, which seems to be the predominant form of the protein in crude brain extract. Cdk5/p35 is the first example of a Cdc2-like kinase with neuronal function.

    Nature 1994;371;6496;423-6

  • p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5.

    Tsai LH, Delalle I, Caviness VS, Chae T and Harlow E

    Massachusetts General Hospital Cancer Center, Charlestown 02129.

    Cyclin-dependent kinase 5 (Cdk5) was originally isolated through its structural homology to human Cdc2, a key regulator of cell-cycle progression. In tissue samples from adult mice, Cdk5 protein is found at the highest level in brain, at an intermediate level in testis, and at low or undetectable levels in all other tissues, but brain is the only tissue that shows Cdk5 histone H1 kinase activity. No equivalent kinase activity has been found in tissue culture cell lines despite high levels of Cdk5. This raised the possibility that a Cdk5 regulatory subunit was responsible for the activation of Cdk5 in brain. Here we describe the cloning and characterization of a regulatory subunit for Cdk5 known as p35. p35 displays a neuronal cell-specific pattern of expression, it associates physically with Cdk5 in vivo and activates the Cdk5 kinase. p35 differs from the mammalian cyclins and thus represents a new type of regulatory subunit for cyclin-dependent kinase activity.

    Nature 1994;371;6496;419-23

  • Chromosomal mapping of human CDK2, CDK4, and CDK5 cell cycle kinase genes.

    Demetrick DJ, Zhang H and Beach DH

    Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, NY 11724.

    Cyclin dependent kinases (CDK's) are kinases that interact with cyclins and regulate cell division. Genomic clones encoding human CDK2, CDK4, and CDK5 were obtained and mapped to their respective chromosomal loci using fluorescence in situ hybridization on human lymphocyte metaphase spreads. Interestingly, CDK2 and CDK4 were located at the same position, 12q13, and CDK5 was mapped to 7q36. 12q13 has been shown to be associated with chromosome alterations such as amplifications and translocations in solid tumors. 7q36 does not appear to be a major site of chromosome alterations in tumors. As CDK2 and CDK4 appear to be important in regulating the human cell cycle, it is possible that the alterations of the 12q13 locus in tumors may involve changes in the regulation of CDK2 and CDK4 genes.

    Funded by: NIGMS NIH HHS: GM 39620

    Cytogenetics and cell genetics 1994;66;1;72-4

  • p42 MAP kinase phosphorylation sites in microtubule-associated protein tau are dephosphorylated by protein phosphatase 2A1. Implications for Alzheimer's disease [corrected].

    Goedert M, Cohen ES, Jakes R and Cohen P

    MRC Laboratory of Molecular Biology, Cambridge, UK.

    The paired helical filament (PHF), which comprises the major fibrous element of the neurofibrillary tangle of Alzheimer's disease, is composed of abnormally phosphorylated microtubule-associated protein tau. Here we show that p42 MAP kinase phosphorylates recombinant tau and converts it to a form which is similar to PHF tau. Of the major serine/threonine protein phosphatases found in mammalian tissues only protein phosphatase 2A (PP2A) could dephosphorylate tau phosphorylated in this manner, with PP2A1 being the most effective form of the enzyme.

    FEBS letters 1992;312;1;95-9

  • A family of human cdc2-related protein kinases.

    Meyerson M, Enders GH, Wu CL, Su LK, Gorka C, Nelson C, Harlow E and Tsai LH

    Massachusetts General Hospital Cancer Center, Charlestown 02129.

    The p34cdc2 protein kinase is known to regulate important transitions in the eukaryotic cell cycle. We have identified 10 human protein kinases based on their structural relation to p34cdc2. Seven of these kinases are novel and the products of five share greater than 50% amino acid sequence identity with p34cdc2. The seven novel genes are broadly expressed in human cell lines and tissues with each displaying some cell type or tissue specificity. The cdk3 gene, like cdc2 and cdk2, can complement cdc28 mutants of Saccharomyces cerevisiae, suggesting that all three of these protein kinases can play roles in the regulation of the mammalian cell cycle. The identification of a large family of cdc2-related kinases opens the possibility of combinatorial regulation of the cell cycle together with the emerging large family of cyclins.

    The EMBO journal 1992;11;8;2909-17

  • Proceedings: Aneurysmal bone cysts of the spine.

    Ameli NO, Teymoorian GA, Saleh H and Slamdoost A

    Acta neurochirurgica 1975;31;3-4;273

Gene lists (6)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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